Use of nernstein voltage sensitive dyes in measuring transmembrane voltage

ABSTRACT

Transmembrane potential measurement methods using cationic dyes, and anionic dyes are provided. Compositions of the cationic and anionic dyes and microfluidic systems which include the dyes and membranes are provided in conjunction with processing elements for transmembrane potential measurements.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a non-provisional of “USE OF NERNSTEINVOLTAGE SENSITIVE DYES IN MEASURING TRANSMEMBRANE POTENTIAL,” U.S. S No.60/158,323, by Farinas and Wada, filed Oct. 8, 1999, and “USE OFNERNSTEIN VOLTAGE SENSITIVE DYES IN MEASURING TRANSMEMBRANE POTENTIAL,”U.S. S No. 60/168,792, by Farinas and Wada, filed Dec. 2, 1999, and “USEOF NERNSTEIN VOLTAGE SENSITIVE DYES IN MEASURING TRANSMEMBRANEPOTENTIAL,” U.S. S No. 60/229,951, by Farinas and Wada, filed Sep. 1,2000. The present application claims priority to and benefit of each ofthese prior applications, pursuant to 35 U.S.C. 119, as well as anyother applicable statute or rule.

COPYRIGHT NOTIFICATION

[0002] Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion ofthis disclosure contains material which is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or patent disclosure, asit appears in the Patent and Trademark Office patent file or records,but otherwise reserves all copyright rights whatsoever.

FIELD OF THE INVENTION

[0003] The present invention is in the field of transmembrane potentialmeasurement using Nernstian dyes, e.g., in microfluidic systems.

BACKGROUND OF THE INVENTION

[0004] Cell-based assays are often preferred for an initial screening ofbiologically active compounds, due to the approximation of in vivosystems by cells, combined with their capability to be rapidly screened.A variety of cell responses to stimuli can be detected, including celldeath, transporter function and response to chemical stimuli.

[0005] The distribution of a permeable ion between the inside andoutside of a cell or vesicle depends on the transmembrane potential ofthe cell membrane. In particular, for ions separated by a semi permeablemembrane, the electrochemical potential difference (Δμ_(j)) which existsacross the membrane, is given by Δμ_(j)=2.3 RT log[j_(l)]/[j_(o)]+zE_(R)F, where R is the universal gas constant, T is anabsolute temperature of the composition, F is Faraday's constant incoulombs, [j_(l)] is the concentration of an ion (j) on an internal orintracellular side of the at least one membrane, [j_(o)] is theconcentration of j on an external or extracellular side of the at leastone membrane, z is a valence of j and E_(R) is a measured transmembranepotential. Thus, the calculated equilibrium potential difference (E_(j))for ion j=−2.3RT(zF)⁻¹log[j_(l)]/[j_(o)] (this is often referred to asthe Nernst equation). See, Selkurt, ed. (1984) Physiology 5^(th)Edition, Chapters 1 and 2, Little, Brown, Boston, Mass. (ISBN0-316-78038-3); Stryer (1995) Biochemistry 4^(th) edition Chapters 11and 12, W. H. Freeman and Company, NY (ISBN 0-7167-2009-4); Haugland(1996) Handbook of Fluorescent Probes and Research Chemicals SixthEdition by Molecular Probes, Inc. (Eugene Oreg.) Chapter 25 (MolecularProbes, 1996) and http://www.probes.com/handbook/sections/2300.html(Chapter 23 of the on-line 1999 version of the Handbook of FluorescentProbes and Research Chemicals Sixth Edition by Molecular Probes, Inc.)(Molecular Probes, 1999) and Hille (1992) Ionic Channels of ExcitableMembranes, second edition, Sinauer Associates Inc. Sunderland, Mass.(ISBN 0-87893-323-9) (Hille), for an introduction to transmembranepotential and the application of the Nernst equation to transmembranepotential. In addition to the Nernst equation, various calculationswhich factor in the membrane permeability of an ion, as well as Ohm'slaw, can be used to further refine the model of transmembrane potentialdifference, such as the “Goldman” or “constant field” equation andGibbs-Donnan equilibrium. See Selkurt, ed. (1984) Physiology 5^(th)Edition, Chapter 1, Little, Brown, Boston, Mass. (ISBN 0-316-78038-3)and Hille at e.g., chapters 10-13.

[0006] Increases and decreases in resting transmembranepotential—referred to as membrane depolarization and hyperpolarization,respectively—play a central role in many physiological processes,including nerve-impulse propagation, muscle contraction, cell signalingand ion-channel gating. Potentiometric optical probes (typicallypotentiometric dyes) provide a tool for measuring transmembranepotential and changes in transmembrane potential over time (e.g.,transmembrane potential responses following the addition of acomposition which affects transmembrane potential) in membranecontaining structures such as organelles (including mitochondria andchloroplasts), cells and in vitro membrane preparations. In conjunctionwith probe imaging techniques (e.g., visualization of the relevantdyes), these probes are employed to map variations in transmembranepotential across excitable cells and perfused organs.

[0007] For example, the plasma membrane of a cell at rest typically hasa transmembrane potential of approximately −20 to −70 mV (negativeinside) as a consequence of K⁺, Na⁺ and Cl⁻ concentration gradients(and, to a lesser extent, H⁺, Ca²⁺, and HCO₃ ⁻) that are maintained byactive transport processes. Potentiometric probes are important toolsfor studying these processes, as well as for visualizing, e.g.,mitochondria (which exhibit a large transmembrane potential ofapproximately −150 mV, negative inside matrix), and for cell viabilityassessment. See, Molecular Probes (1996) chapter 25 and the referencescited therein.

[0008] Potentiometric probes include cationic or zwitterionic styryldyes, cationic rhodamines, anionic oxonols, hybrid oxonols andmerocyanine 540. The class of dye determines factors such asaccumulation in cells, response mechanism and cell toxicity. See,Molecular Probes 1999 and the reference cited therein; Plasek et al.(1996) “Indicators of Transmembrane potential: a Survey of DifferentApproaches to Probe Response Analysis.” J Photochem Photobiol; Loew(1994) “Characterization of Potentiometric Membrane Dyes.” Adv Chem Ser235, 151 (1994); Wu and Cohen (1993) “Fast Multisite Optical Measurementof Transmembrane potential” Fluorescent and Luminescent Probes forBiological Activity, Mason, Ed., pp. 389-404; Loew (1993)“Potentiometric Membrane Dyes.” Fluorescent and Luminescent Probes forBiological Activity, Mason, Ed., pp. 150-160; Smith (1990)“Potential-Sensitive Molecular Probes in Membranes of BioenergeticRelevance.” Biochim Biophys Acta 1016, 1; Gross and Loew (1989)“Fluorescent Indicators of Transmembrane potential:Microspectrofluorometry and Imaging.” Meth Cell Biol 30, 193; Freedmanand Novak (1989) “Optical Measurement of Transmembrane potential inCells, Organelles, and Vesicles” Meth Enzymol 172, 102 (1989); Wilsonand Chused (1985) “Lymphocyte Transmembrane potential and Ca⁺²-SensitivePotassium Channels Described by Oxonol Dye Fluorescence Measurements”Journal of Cellular Physiology 125:72-81; Epps et al. (1993)“Characterization of the Steady State and Dynamic FluorescenceProperties of the Potential Sensitive dye bis-(1.3-dibutylbarbituricacid) trimethine oxonol (DiBAC₄(3) in model systems and cells” Chemistryof Physics and Lipids 69:137-150, and Tanner et al. (1993) “FlowCytometric Analysis of Altered Mononuclear Cell Transmembrane potentialInduced by Cyclosporin” Cytometry 14:59-69.

[0009] Potentiometric dyes are typically divided into at least twocategories based on their response mechanism. The first class of dyes,referred to as fast-response dyes (e.g., styrylpyridinium dyes; see,e.g., Molecular Probes (1999) at Section 23.2), operate by a change inthe electronic structure of the dye, and consequently the fluorescenceproperties of the dye, i.e., in response to a change in an electricfield which surrounds the dye. Optical response of these dyes issufficiently fast to detect transient (millisecond) potential changes inexcitable cells, e.g., isolated neurons, cardiac cells, and even intactbrains. The magnitude of the potential-dependent fluorescence change isoften small; fast-response probes typically show a 2-10% fluorescencechange per 100 mV.

[0010] The second class of dyes, referred to as slow-response orNernstian dyes (See, e.g., Molecular Probes, 1999 at Section 23.3),exhibit potential-dependent changes in membrane distribution that areaccompanied by a fluorescence change. The magnitude of their opticalresponses is typically larger than that of fast-response probes.Slow-response probes, which include cationic carbocyanines, rhodaminesand anionic oxonols, are suitable for detecting changes in a variety oftransmembrane potentials of, e.g., nonexcitable cells caused by avariety of biological phenomena, such as respiratory activity, ionchannel permeability, drug binding and other factors. The structures ofa variety of available slow response dyes are found e.g., at table 25.3of Molecular Probes (1996).

[0011] Many slow, Nernstian dyes such as carbocyanines, rhodamines andoxonols are used to measure transmembrane potential by virtue ofvoltage-dependent dye redistribution and fluorescence changes resultingfrom the redistribution. Fluorescence changes which may be caused byredistribution include: a change of the concentration of the fluorophorewithin the cell or vesicle, a change in the dye fluorescence due toaggregation or a change in dye fluorescence due to binding tointracellular or intravesicular sites. Typically, 10-15 minutes ofequilibration time is used to allow the dyes to redistribute across theplasma membrane after changing the transmembrane potential.

[0012] Despite the availability of transmembrane potential sensorcompositions and assays, there still exists a need for additionalclasses of dyes and for new assays and techniques for usingpotentiometric dyes in biological assays. The present invention fulfillsthese and a variety of other needs which will become apparent uponcomplete review of the following.

SUMMARY OF THE INVENTION

[0013] It is surprisingly discovered that membrane permeable cationicnucleic acid staining dyes can be used as potentiometric dyes formeasuring changes in transmembrane potential. In addition, it wasdiscovered that using both cationic dyes (including, but not limited tomembrane permeable cationic nucleic acid staining dyes) and anionicmembrane permeable redistributing dyes for monitoring changes intransmembrane potential increases the dynamic range and sensitivity oftransmembrane potential measurements. Compositions comprising these twoclasses of dyes and a membrane, as well as microfluidic systems forusing the dyes to measure transmembrane potential, are provided. Furtherit was discovered that measuring the time course of dye uptake, ratherthan equilibrium distributions of the dyes, leads to improvements insignal to noise ratio, speed of the assay and other benefits.

[0014] Accordingly, the present invention provides methods of generatingoptical signals which depend on transmembrane potential or one or morechange in transmembrane potential. For example, in one class ofembodiments, the methods include providing a first component comprisingone or more membrane, adding a cationic membrane permeable nucleic acidstaining dye to the first component, and monitoring a first signaloutput from the cationic membrane permeable nucleic acid staining dye.To monitor changes in transmembrane potential, changes in the firstsignal output are monitored over time. The first signal output is thencorrelated with the transmembrane potential to provide an indication oftransmembrane potential or changes in transmembrane potential.Typically, the first composition is also contacted with an anionicmembrane permeable redistributing dye to increase the sensitivity anddynamic range of the assay.

[0015] In one common format, the relevant components are provided in amicrofluidic system. For example, a method of producing a signal whichis dependent on transmembrane potential is provided, in which a firstmixture which includes one or more membranes and one or more voltagesensitive dyes is flowed through a first channel region. At least afirst signal output is monitored from at least one of the voltagesensitive dyes, thereby producing a signal which is dependent on thetransmembrane potential across the one or more membranes. For example,the voltage sensitive dyes can include one or more membrane permeableredistributing dyes, including one or more ionic dye. The one or moremembrane permeable dyes are typically flowed from a source to the firstchannel region and into contact with the one or more membranes and flowof the membrane permeable labels across the membrane is detected bymonitoring the one or more signal outputs from the membrane permeablelabels, typically before equilibrium is reached. The mixture can includea cationic dye, a cationic membrane permeable nucleic acid staining dye,an anionic dye and/or a neutral dye. The one or more voltage sensitivedyes can include, e.g., an anionic or cationic dye (or both), includingany or all of: Oxonol V, Oxonol VI, DiBAC4(3), DiBAC4(5), DiBAC2(3), acationic dye, a cationic membrane permeable nucleic acid staining dye,and a SYTQ dye such as SYTO 62.

[0016] For example, a cationic dye, such as the cationic membranepermeable nucleic acid staining dye, and the first component are flowedthrough at least a first microfluidic channel comprising flowing thecationic membrane permeable nucleic acid staining dye through the firstor second microchannel and into contact with the at least one membrane.An anionic membrane permeable redistributing dye can also be flowedthrough the first or second channel and into contact with the at leastone membrane.

[0017] Thus, in microfluidic formats, methods of measuring or monitoringchanges in a transmembrane potential are provided. In the methods, afirst component which includes one or more membrane is flowed from asource to a first channel region. A labeling composition comprising amembrane permeable label is flowed into contact with the membrane. Themembrane is altered in some way that causes an alteration intransmembrane potential, e.g., by changing the ionic composition on oneside of the membrane (e.g., inside or outside of a cell) or by changingthe permeability of the membrane to ions. The flow of the membranepermeable label across the membrane is monitored by monitoring a firstsignal output from the membrane permeable label, thereby measuringchanges in the transmembrane potential.

[0018] The above methods can include contacting the membrane to one ormore transmembrane potential modulatory compositions and monitoring aneffect of the one or more transmembrane potential modulatorycompositions on the transmembrane potential (e.g., by monitoring thefirst signal), thereby monitoring an effect of the one or moretransmembrane potential modulatory compositions on the transmembranepotential. This can be used as a drug screening method for testingpotential modulatory compounds for a transmembrane potential modulatoryactivity. Examples of modulatory compositions include hyperpolarizationbuffers, depolarization buffers, compounds which alter the ionicpermeability of a membrane, and the like. In addition, controlmodulators (modulators having a known effect on transmembrane potentialin the relevant assay) can be compared to test modulators having unknowneffects to determine membrane modulatory activity of the testmodulators. Dose response curves for either control or test modulatorscan be determined and the curves compared.

[0019] Control and test modulators can affect, e.g., transporteractivity, ion channel activity, or other factors which have an effect ontransmembrane potential and changes in transmembrane potential. Examplesof test and control modulators include a variety of compounds whicheffect membrane ionic permeability, ionic potential or the like,including neurotoxins (e.g., such as palytoxin), sets of neurotoxins,neurotransmitters, sets of neurotransmitters, proteins, sets ofproteins, peptides, sets of peptides, lipids, sets of lipids,carbohydrates, sets of carbohydrates, organic molecules, sets of organicmolecules, drugs, sets of drugs, receptor ligands, sets of receptorligands, antibodies, set of antibodies, cytokines, sets of cytokines,chemokines, sets of chemokines, hormones, sets of hormones, cells, setsof cells and the like.

[0020] In general, the time course of dye translocation across themembrane depends on the transmembrane potential across the membrane.Thus, at a selected time (t) after adding a dye to a membrane, theamount of signal from the dye is correlated to transmembrane potentials.Typically, (t) can be less than about 100 seconds. Commonly, (t) isbetween about 0.1 and 80 seconds, e.g., between about 10 and 70 seconds.A ratio of first and second signals from the cationic and anionic dyesnoted above (e.g., over time) can be determined to further refineestimates of changes in transmembrane potential.

[0021] Examples of useful dyes include cyclic-substituted unsymmetricalcyanine dyes and other cationic membrane permeable nucleic acid stains.Examples of useful dyes include Blue-fluorescent SYTO dyes,Green-fluorescent SYTO Dyes, Orange-fluorescent SYTO dyes,Red-fluorescent SYTO dyes such as SYTO 62, Pur-1, thiazol, aryl,2DS-7J1, Hoechst 33258, Hoechst 33342 and hexidium iodide. Commonanionic membrane permeable redistributing dyes include anionicbis-isoxazolone oxonol dyes, bis-oxonol dyes and others. For example,the anionic membrane permeable redistributing dye can be e.g., Oxonol V,Oxonol VI, DiBAC₄(3), DiBAC₄(5) and/or DiBAC₂(3). Example dyeconcentrations in the relevant systems are typically between about 0.01and about 50 μM. For example, the cationic dye can be SYTO 62, added tothe first component to a concentration of between about 0.01 and about50 μM and the anionic dye can be DiBAC4(3), added to the first or secondcomponent at a concentration of between about 0.01 and about 50 μM.

[0022] As noted, examples of membranes of interest include cells,organelles, artificial membranes, membrane preparations of artificial ornaturally occurring membrane sources, and the like. Membranepreparations can be suspended in any suitable buffer, e.g., a fluidcomprising a membrane permeable ion such as Na⁺, K⁺, Cl⁻, H⁺, Ca²⁺, orHCO₃ ⁻. In one embodiment, the membrane is present in an intact or livecell such as an animal cell, a plant cell, a fungal cell, a bacterialcell, or the like. For example, the cell can be a mammalian cell such asa primate cell, a rodent cell, a canine cell, a feline cell, or alivestock cell, or can be e.g., an insect cell or other animal cell. Thecell can be a cultured cell such as a THP-1 cell, a COS cell, a CHOcell, a HEK cell, a jurkat cell, a βRL cell, a HeLA cell, an NIH 3T3cell, an RBL-2H3 cell, or the like. The cell can also be a primary cellsuch as a cell isolated from endoderm, ectoderm, mesoderm,differentiated tissue, undifferentiated tissue, partially differentiatedtissue, blood, peripheral blood, nerve, muscle, skin, bone, or the like.

[0023] Typically, signal outputs from dyes are detected by monitoringone or more fluorescent emission produced by the relevant dye. This canbe performed spectrophotometrically, optically or, e.g., via microscopy.

[0024] In one aspect, the invention provides a microfluidic device formonitoring transmembrane potential. The microfluidic device includes abody structure having at least one microscale cavity (e.g.,microchannel, microchamber, or the like) disposed therein. A targetsource of a first composition which includes at least one membrane isfluidly coupled to the at least one microscale cavity (e.g., amicroscale channel, chamber, well, column or the like). A cationicmembrane permeable staining dye source which includes one or morecationic membrane permeable nucleic acid staining dye, is fluidlycoupled to the at least one microscale cavity. Alternatively or inaddition, an anionic membrane permeable redistributing dye source whichincludes one or more anionic redistributing dye is fluidly coupled tothe at least one microscale cavity. During operation of the device, thefirst composition is contacted, in the presence of the cationic membranepermeable staining dye, and/or the anionic membrane permeableredistributing dye, to at least one transmembrane potential modulatorycomposition.

[0025] In applications where the device is used for screening effects ofmodulatory compositions, the device can include a source of at least onepotential membrane modulatory composition fluidly coupled to the atleast one microscale cavity. The potential membrane modulatorycomposition can be, e.g., a membrane hyperpolarization buffer, amembrane depolarization buffer, or a compound which alters ionicpermeability of the membrane.

[0026] The device typically includes a signal detector located proximalto the microscale cavity. The signal detector detects the detectablesignal, e.g., for a selected length of time (t). For example, thedetector can include a spectrophotometer, or an optical detectionelement. Commonly, the signal detector is operably coupled to acomputer, which deconvolves the detectable signal to provide anindication of the transmembrane potential, e.g., an indication of achange in the potential over time.

[0027] In one typical embodiment, during operation of the device, thefirst composition comprising at least one membrane is flowed from thetarget source into the cavity, e.g., into a microchannel. The potentialmembrane modulatory composition is flowed from the target source intocontact with the first composition. The cationic membrane permeablestaining dye and/or an anionic membrane permeable redistributing dye isflowed into contact with the first composition and the detectable signalis monitored at a selected time (t) after contact of the firstcomposition with the cationic membrane permeable staining dye and/or thean anionic membrane permeable redistributing dye.

[0028] In one aspect, the invention provides a composition, e.g., forpracticing the methods noted above. The composition includes a firstcomponent comprising a membrane, a cationic membrane permeable nucleicacid staining dye, and an anionic membrane permeable redistributing dye.The membrane component can include, e.g., a cell, mitochondria,chloroplast, cell vesicle, a membrane preparation of a cell or cellcomponent, or an artificial membrane. The cell can be an intact cellwhich can be, e.g., an animal cell, a plant cell, a fungal cell or abacterial cell. For example, the cell can be any of those noted herein.

[0029] Similarly, the cationic membrane permeable nucleic acid stainingdye and the anionic membrane permeable redistributing dye can be any ofthose noted above with reference to the methods of the invention. Thecomposition can also include buffers, ions, etc., as noted herein. Acontainer or microfluidic processor comprising the composition is also afeature of the invention. For example, the composition of the inventioncan be present in a kit or microfluidic processor. The kit canadditionally include, e.g., instructions for practicing the method ofthe invention, control compounds, test compounds, containers for holdingreagents, packaging materials, or the like.

BRIEF DESCRIPTION ON THE FIGURES

[0030]FIG. 1 is a line graph showing the time course of dye uptake as afunction of transmembrane potential. Line A shows resting potential,high sodium. Line B shows depolarized, high potassium.

[0031]FIG. 2 is a line graph showing change in SYTO 62 fluorescence overtime. Line A shows resting potential, high sodium. Line B showsdepolarized, high potassium.

[0032]FIG. 3, panel A is a bar graph showing fluorescence ratio ofDiBAC₄(3) to SYTO® 62. Panel B shows two graphs of fraction of cells vs.DiBAC₄(3) intensity and SYTO 62 intensity. Line A shows resting cells inhigh sodium buffer, line B shows depolarized cells in UTP/high potassiumbuffer and line C shows hyperpolarized cells in UTP/high sodium buffer.

[0033]FIG. 4 is a bar graph of a dose-response curve.

[0034]FIG. 5 is a schematic of the Caliper® 3B5 microfluidic processor.

[0035]FIG. 6 is a schematic of an integrated system of the invention.

[0036]FIG. 7 is a schematic showing further details of an integratedsystem of the invention.

[0037]FIG. 8 is a schematic showing further details of an integratedsystem of the invention.

[0038]FIG. 9, panels 1-D schematically depicts a membrane potentialassay and shows data from the assays.

[0039]FIG. 10, panels A-C schematically shows depolarization andhyperpolarization assays.

[0040]FIG. 11 is a data figure schematically showing, e.g., low cellconsumption and high data quality during use of primary cells in assaysof the invention.

[0041]FIG. 12 is a calibration curve showing the ratio of dibac/syto 62versus membrane potential.

DETAILED DESCRIPTION OF THE INVENTION

[0042] The distribution of a permeable ion between the inside andoutside of a cell or vesicle, or between the inner and outer leaflet ofa membrane, depends on transmembrane potential. The voltage dependenceof the distribution is essentially governed by the Nernst equation. Manyslow Nernstian dyes such as carbocyanines, rhodamines and oxonols areused to measure transmembrane potential by virtue of voltage-dependentdye redistribution and fluorescence changes resulting from theredistribution. Fluorescence changes which are caused by redistributioninclude: a change of the concentration of the fluorophore within thecell or vesicle, a change in the dye fluorescence due to aggregation ora change in dye fluorescence due to binding to intracellular orintravesicular solutes. Typically, 10-15 minutes of equilibration timeis used to allow the dyes to redistribute across the plasma membraneafter changing the transmembrane potential (TMP).

[0043] The present invention concerns a class of dyes which are newlydiscovered to be suitable for generating optical signals which depend onTMP. The class of dyes are cationic, membrane permeable, nucleic acidstains such as SYTO 62. The invention also relates to the method inwhich they are used to measure changes in TMP. After addition of SYTO 62to cell suspensions, a time course of SYTO 62 fluorescence was found tobe dependent on the transmembrane potential across the cell plasmamembrane. A significant advantage of this new class of probes comparedtogether cationic probes is that a large fraction of the fluorescencechange arises from fluorophore located outside of mitochondria. Theelectromotive force on these cationic dyes is also counter to anionicdyes, leading to important advantages when the two types of dyes areused together in TMP assays.

[0044] A preferred method for using this class of dyes is to use the newclass of dyes together with a traditional anionic Nemstian dye such asDIBAC₄(3) and to measure the ratio of their fluorescence intensities.This approach has several advantages, such as a higher signal to noiseratio than when the dyes are used separately. Another advantage is theability to detect voltage changes involving both hyperpolarization anddepolarization more efficiently, because depolarization enhances anionicdye fluorescence (decreasing cationic dye fluorescence) andhyperpolarization enhances cationic dye fluorescence (decreasing anionicdye fluorescence).

[0045] A further preferred method is to measure cell-associatedfluorescence of the dyes after contacting a cell with the dyes, butbefore an equilibrium distribution of the dyes has been established.This kinetic approach increases sensitivity, provides larger dynamicrange to the assay, and allows measurements to be made more quickly at agiven sensitivity level. This approach has operational advantages suchas eliminating manual preload of cells with dyes that can be a source of“run to run” and “within run” assay variation or cellular toxicity. Thismethod is also highly suited to microfluidic processor formats thatallow an automated addition of test samples and dyes, followed by abrief incubation and reading of one or two color fluorescence.

[0046] The specific chemistry of the cationic nucleic acid (e.g., DNAand/or RNA) stain can be varied, as can the specific chemistry of theanionic Nernstian dye. The methods can utilize microfluidicinstrumentation or more traditional assay formats such as fluorescencemicroplate readers with multiple reagent addition capability.

[0047] Definitions

[0048] A “transmembrane potential” is the work needed to move a unit ofcharge across a membrane.

[0049] A “cationic membrane permeable nucleic acid staining dye” is adye which has a positive charge under specified pH (e.g., physiologicalpH) and which binds to or otherwise associates with a nucleic acid,where the dye can cross a selected membrane such as the membrane of anintact cell.

[0050] An “anionic Nernstian dye” or an anionic membrane permeableredistributing dye is a dye which has a negative charge at a specifiedpH (e.g., physiological pH) and which is membrane permeable and whosedistribution between the inside and outside of the space bounded by themembrane or between the inside and outside of the membrane, depends onthe transmembrane potential across the membrane.

[0051] A “neutral dye” has an overall neutral charge under the relevantconditions at issue, e.g., a specified pH (e.g., physiological pH).

[0052] A “voltage sensing composition” is a transmembrane potentialindicator comprising a fluorescent dye. Common voltage sensingcompositions of the invention include one or more cationic membranepermeable nucleic acid staining dye(s), or, optionally, one or moreadditional cationic potentiometric dye, and, optionally, an anionicmembrane permeable redistributing dye.

[0053] A membrane is “depolarized” when the transmembrane potentialacross the membrane is zero. A membrane is “hyperpolarized” when thetransmembrane potential is more negative than the resting potential ofthe membrane.

[0054] A membrane is “permeable” to a given component (dye, ion, etc.)when that component can equilibrate across the membrane in about 24hours or less, and generally within about 12, 5, 1 or 0.5 hours, orless. Permeability can be dependent upon the relevant conditions, e.g.,temperature, ionic conditions, voltage potentials, or the like.

[0055] Membrane Permeable Cationic Dyes

[0056] As noted, preferred voltage sensing compositions of the inventioninclude any of a variety of cationic membrane permeable nucleic acidstaining dyes. Such dyes are available and can be used in the assays,compositions and devices of the invention.

[0057] A feature of the invention is the discovery that cell-permeantnucleic acid stains are suitable for use in potentiometric TMPmeasurements. For example, cyanine nucleic acid-staining SYTO dyesavailable from Molecular Probes (See, e.g., Molecular Probes 1999,Chapter 8). These dyes are generally suitable for use in potentiometricTMP measurements. These include the SYTO orange fluorescent nucleic acidstains (e.g., SYTO 80-85); the SYTO blue fluorescent nucleic acid stains(SYTO 40-45); the SYTO green fluorescent nucleic acid stains (SYTO11-16, 18, and 20-25) and the SYTO red fluorescent Nucleic Acid Stains(SYTO 17, and 59-64). See, Molecular Probes Product information SheetsMP 11360, MP 11350, MP 07572 and MP 11340, respectively and thereferences cited therein.

[0058] The numerous SYTO dyes are nucleic acid stains that passivelydiffuse through the membranes of most cells. These cell-permeant, UV- orvisible light-excitable dyes have been traditionally used to stain RNAand DNA in live and dead eukaryotic cells, as well as in gram-positiveand gram-negative bacteria. These dyes share several characteristics,including permeability to virtually all cell membranes, includingmammalian cells and bacteria; high molar absorptivity, with extinctioncoefficients >50,000 cm⁻¹M⁻¹ at visible absorption maxima; and lowintrinsic fluorescence, with quantum yields typically <0.01. As noted,the dyes are available as blue-, green-, orange- or red-fluorescentdyes. The SYTO dyes differ from each other in one or morecharacteristic(s), including cell permeability, fluorescence enhancementupon binding nucleic acids, excitation and emission spectra, and DNAIRNAselectivity and binding affinity (See, Molecular Probes, 1999, Table8.7). The SYTO dyes are compatible with a variety of fluorescence-basedinstruments that use laser excitation or conventional broadbandillumination sources (e.g., mercury- and xenon-arc lamps).

[0059] The cyanine dyes show differences in some physicalcharacteristics, such as permeability and nucleic acid specificity, thatallow their distribution into distinct classes. In addition to the SYTOdyes, Hoechst 33258 and Hoechst 33342 dyes and others are also useful inthe context of the present invention.

[0060] The recommended dye concentration for cell staining depends onthe assay and may vary widely but is typically 0.1-50 μM for bacteria,0.1-100 μM for yeast and 10 nM-50 μM for other eukaryotes.

[0061] Preferred voltage sensing compositions of the inventionoptionally include any of a variety of other membrane-permeant nucleicacid staining dyes. For example, hexidium iodide reagents (MolecularProbes Catalogue number H-7593) are moderately lipophilicphenanthridinium dyes that are permeant to mammalian cells andselectively stain almost all gram-positive bacteria in the presence ofgram-negative bacteria. Generally, both the cytoplasm and nuclei ofeukaryotic cells show staining with hexidium iodide; however,mitochondria and nucleoli can also be stained.

[0062] Similarly, dihydroethidium is a chemically reduced ethidiumderivative that is permeant to live cells. Dihydroethidium exhibits bluefluorescence in the cytoplasm. Many viable cells oxidize the probe toethidium, which then fluoresces red upon DNA intercalation.

[0063] LDS 751 (Molecular Probes (1999) catalogue L-7595) is acell-permeant nucleic acid stain. LDS 751, which has its peak excitationat ˜543 nm on dsDNA, can be excited by an argon-ion laser at 488 nm andis useful in multicolor analyses due to its long-wavelength emissionmaximum (˜712 nm).

[0064] ACMA (9-amino-6-chloro-2-methoxyacridine, A-1324) is a DNAintercalator that selectively binds to poly(d(A-T)). ACMA binds tomembranes in the energized state and becomes quenched if a pH gradientforms. ACMA can be used in the present invention or excluded from use.For example, in one embodiment, a nucleic acid stain other than ACMA isused for potentiometric measurements. ACMA can also be used inpotentiometric measurements.

[0065] In addition, classes of cationic membrane permeable dyes otherthan nucleic acid stains can be used in the voltage sensing compositionsof the invention, e.g., in conjunction with the cationic membranepermeable nucleic acid staining dyes noted above, and/or in combinationwith anionic dyes in non-equilibrium measurements of changes in TMP, orin other applications as noted herein. Such dyes include, e.g.,indo-carbocyanine dyes, thio-carbocyanine dyes, oxa-carbocyanine dyes(see Molecular Probes on-line catalogue, updated as of Aug. 10, 2000, atsection 23.3, entitled “Slow-Response Dyes;”http://www.probes.com/handbook/sections/2303.html). See also, Sims, etal. (1974) “Studies on the Mechanism by Which Cyanine Dyes MeasureMembrane Potential in Red Blood Cells and Phosphatidylcholine Vesicles,”Biochemistry 13, 3315; Cabrini and Verkman (1986) “Potential-SensitiveResponse Mechanism of DiS-C3(5) in Biological Membranes,” Membrane Biol92, 171; Guillet and Kimmich (1981) “DiO-C3(5) and DiS-C3-(5):Interactions with RBC, Ghosts and Phospholipid Vesicles,” J MembraneBiol 59, 1; Rottenberg and Wu (1998) “Quantitative Assay by FlowCytometry of the Mitochondrial Membrane Potential in Intact Cells,”Biochim Biophys Acta 1404, 393 (1998).

[0066] Other useful dyes include amino napthylethylenyl pyridinium dyes,and dialkyl amino phenyl polyphenyl pyridinium dyes. The aminonapthylethylenyl pyridinium dyes include the ANEP type dyes, e.g.,listed in the Molecular Probes catalog (Di-4-ANEPPS, Di-8-ANEPPS,Di-2-ANEPEQ, Di-8-ANEPEQ and Di-12-ANEPEQ). Dialkyl amino phenylpolyphenyl pyridinium dyes include the RH type dyes listed in theMolecular Probes catalog (RH160, RH237, RH 421, RH 704, RH 414, and RH461).

[0067] Another class of cationic probes which can be used with thecationic membrane permeable nucleic acid staining dyes of the invention,or in combination with anionic dyes of the invention, e.g., innon-equilibrium measurements of changes in TMP, or in other applicationsas noted herein, are rhodamine probes. Rhodamine 123 (Molecular Probes1999 catalogue number R-302) is widely used as a structural marker formitochondria and as an indicator of mitochondrial activity. The methyland ethyl esters of tetramethylrhodamine are in use as dyes fordetermining transmembrane potential by quantitative imaging.Quantitative transmembrane potential measurements utilizing the Nernstequation imply that the membrane distribution of the dye depends on thetransmembrane potential and that other processes, such as dyeaggregation and potential-independent interactions with intracellularcomponents, contribute minimally. The methyl and ethyl esters oftetramethylrhodamine, e.g., TMRM and TMRE fulfill these criteria (See,Molecular Probes, 1999, supra). They are more membrane-permeant thanrhodamine 123, and their strong fluorescence means that they can be usedat low concentrations, thus avoiding aggregation. Because theirfluorescence is relatively insensitive to the environment, spatiallyresolved fluorescence of TMRM and TMRE presents an unbiased profile ofmembrane distribution that can be directly related to transmembranepotential via the Nernst equation. This is particularly useful inembodiments where theses cationic dyes are used in conjunction withanionic dyes in monitoring changes in TMP under non-equilibrium dyedistribution conditions.

[0068] Anionic Membrane Permeable Redistributing Dyes

[0069] As noted, preferred voltage sensing compositions of the inventioninclude any of a variety of anionic membrane permeable redistributingdyes, including which are available and can be used in the assays,compositions and devices of the invention. Additional new anionic dyeswhich developed which are membrane permeable can also be used in themethods and systems herein.

[0070] Examples of available anionic dyes include the anionicbis-isoxazolone oxonols which accumulate in the cytoplasm of depolarizedcells by a Nernst equilibrium-dependent uptake from the extracellularsolution. Of the oxonols studied in one reference (“Kinetics of thePotential-Sensitive Extrinsic Probe Oxonol VI in Beef HeartSubmitochondrial Particles.” J. C. Smith, B. Chance. J Membrane Biol 46,255 (1979)), oxonol VI gave the largest spectral shifts, with anisosbestic point at 603 nm. Oxonol VI responds to changes in potentialmore rapidly than oxonol V.

[0071] The three common bis-barbituric acid oxonols, often referred toas DiBAC dyes, form a family of spectrally distinct potentiometricprobes with excitation maxima at approximately 490 nm (DiBAC₄(3), 530 nm(DiSBAC₂(3)) and 590 nm (DiBAC₄(5)). DiBAC₄(3) has been used in manypublications that cite using a “bis-oxonol” (Molecular Probes, 1999,chapter 23). The dyes enter depolarized cells where they bind tointracellular proteins or membranes and exhibit enhanced fluorescenceand red spectral shifts. Increased depolarization results in more influxof the anionic dye and thus an increase in fluorescence. DiBAC₄(3) hasparticularly high voltage sensitivity. The long-wavelength DiSBAC₂(3)has frequently been used in combination with the UV light-excitable Ca²⁺indicators indo-1 or fura-2 for the simultaneous measurements oftransmembrane potential and Ca²⁺ concentrations (id. at Table 23.2).

[0072] Non-Equilibrium TMP Change Measurements: Cationic, Anionic andNeutral Dye Compositions

[0073] One aspect of the present invention is the surprising discoverythat measurement of dye uptake prior to equilibration of the dye acrossa membrane results in an accurate way of measuring changes in TMP. Inparticular, detection of both cationic and anionic dyes at one or moretime points prior to equilibration of dye across a membrane increasesthe signal to noise and dynamic range for measuring changes in TMP.Often, a single time point measurement is sufficient to provide anindication of changes in TMP.

[0074] Thus, a first component comprising one or more membrane is mixedwith at least a first membrane permeable redistributing dye and one ormore signal output from the first redistributing dye measured before anequilibrium dye distribution is established, providing a non-equilibriumdye distribution measurement. This non-equilibrium dye distributionmeasurement depends on the change in transmembrane potential. Generally,at least a second membrane permeable redistributing dye is added to theone or more component and one or more signal outputs from the secondmembrane permeable redistributing dye is also measured before anequilibrium dye distribution is established. A first signal output fromthe cationic dye and a second signal from the anionic dye is measured atone or more time point, thereby providing an indication of at least onechange in transmembrane potential.

[0075] Any of the membrane permeable nucleic acid staining dyes notedabove are suitable for measuring changes in TMP by the methods herein.In addition, other cationic and/or anionic membrane redistributing dyessuch as cationic rhodamines and the other cationic dyes noted herein canbe used in the methods of the invention, as well as DiBAC₄(3) and theother dyes noted herein. In applications using both cationic and anionicdyes, any of those cationic or anionic dyes noted above can be used togenerate a signal which depends on TMP. It is expected that one of skillcan select optimal dye combinations simply by performing any TMP assaynoted herein using the various dyes which are noted. For example, thecombination of SYTO®62 and DiBAC₄(3) in non-equilibrium TMP assays wasfound to provide for an extended dynamic range for monitoring changes inthe TMP assay and to provide a high signal to noise ratio in the assays.

[0076] The anionic and cationic dyes can be pre-mixed and added to amembrane preparation, e.g., in a microfluidic system, or the dyes can beseparately added to the membrane preparation. The decision whether topre-mix the dyes depends on, e.g., the source of the dyes, thepreference of the user, the type of device in use, and the compatibilityof the dyes to be mixed. In an alternate embodiment, anionic andcationic dyes are used in separate assays, on the same or separatemembrane preparations, with signal measurements from anionic andcationic dyes being taken at similar or different time points and thedata combined by any mathematical method to produce combinatorial datasets, graphs, or the like. Thus, dyes can be combined prior tocontacting membrane preparations, or can be independently contacted tothe same or different membrane preparations. Data sets produced fromsignal measurements from distinct dyes can be analyzed independently orcan be combined.

[0077] Neutral dyes can be used in the methods and as a feature of thecompositions of the invention. In particular, neutral dyes are usefulcontrols in many of the methods herein. For example, a neutral dye canbe used to monitor membrane permeability, including changes inpermeability which are not voltage dependent. For example, the effectsof temperature, pH, solvents, or the like on permeability can bemonitored using a neutral dye, and the changes in permeability due totemperature effects can be used in normalizing or otherwise interpretingdata obtained for cationic and/or anionic dyes. Similarly, a cell orother membrane containing structure can be monitored for being intact bymonitoring a signal output from the neutral dye. In general, neutraldyes are selected such that they do not substantially interfere withother components of the assay (cationic or anionic dyes, membranes,etc.).

[0078] In view of the use of neutral dyes in the assays as set forthherein, the various microscale devices herein can also include a sourceof a neutral dye, means for flowing the dye into contact with membranepreparations and the like.

[0079] A variety of neutral dyes are set forth in the references herein,including Molecular Probes (1999). Such dyes can be polar or non-polar,including neutral red, nonpolar pyrene probes, non-polar BODIPY probes,Nile Red, amphiphilic derivatives of various rhodamines, fluoresceinsand coumarins and many others.

[0080] Membrane Preparations

[0081] One of skill can adapt available cells, membrane preparations andother reagents to the present invention by providing the cells, membranepreparations and other reagents to the microfluidic systems herein asnoted. Assay conditions and buffer and reagent parameters utilizingdyes, modulators, cells, membrane preparations and the like can beselected based upon established TMP activity levels, known pairings ofreagents, concentrations and kinetic information for known TMPmodulators, modified by the addition of a selected modulator or putativemodulator to the system. Initial screens of a particular modulator orputative modulator can be conducted at a single concentration in thesystem, or at multiple modulator concentrations. Typically, compoundswhich have modulatory activity based upon an initial screen are titratedinto contact with the membrane, in increasing or decreasing amounts, toestablish a dose-response curve for the modulator.

[0082] Rather than simply using cells which naturally display TMPresponse to modulators under some set of conditions, recombinant cellscan also be constructed which incorporate desired TMP responseactivities. This is advantageous because certain cells can be easilymaintained in culture using established methods.

[0083] In general, methods of making recombinant cells and expressingcellular proteins such as membrane channel proteins and transporterreceptors are well known in the art. For an introduction to recombinantmethods, see, Berger and Kimmel, Guide to Molecular Cloning Techniques,Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif.(Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual (2nded.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1989 (“Sambrook”); and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”).

[0084] Furthermore, the culture of cell lines and cultured primary cellsfrom tissue or blood samples is well known in the art. Freshney (Cultureof Animal Cells, a Manual of Basic Technique, third edition Wiley-Liss,New York (1994)) and the references cited therein provides a generalguide to the culture of animal cells. The culture of Mammalian Cells isdescribed in Freshney, id., and in Doyle and Griffiths (1997) MammalianCell Culture: Essential Techniques John Wiley and Sons, NY. The cultureof plant cells is described in Payne et al. (1992) Plant cell and tissueculture in liquid systems John Wiley & Sons, Inc. New York, N.Y.Additional information on cell culture, including prokaryotic cellculture, is found in Ausubel, Sambrook and Berger, supra. Cell culturemedia are described in Atlas and Parks (eds) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla. Additionalinformation is found in commercial literature such as the Life ScienceResearch Cell Culture catalogue (1998) from SigmaAldrich, Inc (St Louis,Mo.) and, e.g., the Plant Culture Catalogue and supplement (1997) alsofrom Sigma-Aldrich, Inc (St Louis, Mo.). Many cells can be tested forTMP response to a selected modulator. In the context of the invention,cells which can be tested include those which have cell walls and/orcell membranes which are permeable to the dyes being used.

[0085] Ionic channels are pores in membranes (e.g., cell membranes)which mediate passage of many ions across the relevant membrane. Hille(1992) Ionic Channels of Excitable Membranes, second edition, SinauerAssociates Inc. Sunderland, Mass. (ISBN 0-87893-323-9) (Hille) providean introduction to ionic channels. Example channel types are those whichpermit NA⁺, K⁺, Ca⁺⁺ or other ions to pass through the membrane. Detailsregarding the structure and function of these and other ion channels isfound in Hille. Membrane preparation techniques comprising ion channelsare found in Hille and the references cited therein.

[0086] Another set of proteins which can affect TMP responses aretransporter proteins. These proteins actively transport ions and othermolecules into or out of cells details regarding the cloning andexpression of transporters is found in Neurotransmitter Transporters:Structure, Function and Regulation (1997) M. E. A Reith, ed. HumanPress, Towata N.J.; Neurotransmitter Methods: Methods in MolecularBiology Volume 72 (1997) R. Rayne, ed. Human Press, Towata, N.J.;Neuropeptide Protocols: Methods in Molecular Biology Volume 73: Irvineand Williams, eds., Human Press, Towata N.J.; Neurochemistry: APractical Approach, 2^(nd) edtion (1997) Turner and Bachelard, eds.,Oxford Press, Oxford England; and, Neural Cell Culture: A PracticalApproach (1996) Cohen and Wilkins, eds. Oxford Press, Oxford England.

[0087] The above references also provide a number of membranepreparation protocols for producing a variety of membrane preparationsuseful in the present invention. These include cell preparations,liposome preparations and the like. A variety of additional referencesteach a variety of such membrane preparatory techniques, e.g., Grahamand Higgins (1997) Membrane Analysis Bios Scientific Publishers, Oxford,England; Gould (Ed) (1994) Membrane Protein Expression Systems: A User'sGuide Portland Press, London, England; Gunstone (1996) Fatty Acid AndLipid Chemistry Blackie Academic and Professional, London, England;Yehuda and Mostofsky (Eds.) (1997) Handbook of Essential Fatty AcidBiology: Biochemistry, Physiology and Behavioral Neurobiology HumanaPress, Towata, N.J.; Riafai and Warnick (1994) Laboratory Measurement ofLipids, Lipoproteins and Apolipoproteins AACC Press, Washington, D.C.,and New (Ed) (1990) Liposomes: A Practical Approach IRL Press at Oxford,England.

[0088] TMP Measurments

[0089] Potentiometric measurements are made over time to determinechanges in TMP over time, e.g., in response to TMP modulators. Themeasurements are made by monitoring, e.g., changes in fluorescence overtime. Several distinct emission wavelengths can be monitoredsimultaneously. As discussed below, a variety of microfluidic systemsincorporate fluorescence detectors which can be used in the context ofthe present invention. In addition, fluorescence can be monitored instandard cuvettes and/or microtiter plates, using spectrophotometers andplate readers common in the art.

[0090] Ordinarily, changes in fluorescence are monitored and correlatedto changes in TMP. However, static measurements (e.g., single time pointmeasurements) can also be made and correlated to expected fluorescencemeasurements (e.g., compared to collected (e.g., tabulated) fluorescenceinformation) e.g., for calibration purposes.

[0091] TMP Modulators

[0092] A variety of ionic channel activity modulators are known. Forexample many neurotoxins block specific ion channels, and/or modify thekinetics of channel gating. See, Hille at chapter 17. For example,treatment of cells with pronase results in slowing or loss of Na⁺inactivation.

[0093] A variety of agents which modify gating in NA⁺ and other ionchannels are known and available, such as those listed in Table 1 ofHille at chapter 17. Examples include chemical agents which eliminateTMP inactivation such as pronase, trypsin, NBA, NBS, TNBS, SITS, IO₃,trinitrophenol, Glyoxal, tannic acid, Formaldehyde, glutaraldehyde,pH_(i)<6, pH_(i), >9, Acridine orange, eosine Y plus light andDPI-201-106. Other examples include Scorpion and coelenterate peptide αtoxins slowing membrane inactivation such as those from Leiurusquinquestriatus (North African), Buthus eupeus; B. tamalus (Asian),Androctonus australis (North African), Centruroides scuilpturatus; C.suffusus (North American), Tityus serrulatus (South American), Anemoniasulcata (Mediterranean), Anthopleura xanthogrammica (California), andCondylactis gigantea (Bermuda). Other examples include Scorpion peptideβ toxins shifting membrane activation such as Centruroides sculpturatus,C. suffusus and Tityus serrulatus. Other examples include lipid-solubletoxins shifting activation and slowing inactivation such as Aconitine,veratridine, and batrachotoxin. Similarly, Pyrethroids such asallethrin, dieldrin, aldrin, and tetramethrin have an effect on TMPand/or alterations in TMP. Similarly, grayanotoxins, DDT and analogsaffect TMP and changes in TMP. A variety of ionic conditions affectvoltage dependence of gating, such as the presence of external divalentions and pH, external and internal monovalent ions, and the presence ofcharged or dipolar adsorbants such as lyotropic anions, salicylates andphlorizin.

[0094] Many molecules can be tested for TMP modulatory activity usingthe methods herein. Preferably, the molecules which are tested do notinteract directly with the dyes used in the assays, as any suchinteraction complicates interpretation of any results which areobserved. Similarly, a variety of chemical compounds can be used aspotential modulators in the assays of the invention, although, mostoften, the compounds do not interact directly with the dyes used in theassays and, commonly, compounds which can be dissolved in aqueous ororganic (e.g., DMSO-based) solutions are used to facilitate flow, e.g.,where the assays are conducted in microscale systems. The assays hereinare designed to optionally screen large chemical libraries by automatingthe assay steps and providing compounds from any convenient source tothe assays. It will be appreciated that there are many suppliers ofchemical and biological compounds, including Sigma (St. Louis, Mo.),Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), FlukaChemika-Biochemica Analytika (Buchs Switzerland) and the like.

[0095] In one preferred embodiment, high throughput screening methodsinvolve providing a combinatorial library containing a large number ofpotential TMP activity modulator compounds (“potential modulatorcompounds”). Such “combinatorial chemical libraries” are screened in oneor more assays, as described herein, to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve asconventional “lead compounds” or can themselves be used as potential oractual therapeutics for treating conditions amenable to treatment bymodulating TMP activities. For example, a variety of diseases aretreated by administering TMP modulators, such as transport modulators,including, e.g.,: panic, stress, obsessive compulsive disorders,depression, chronic pain and many other physical and psychologicalconditions. See, Neurotransmitter Transporters: Structure, Function andRegulation (1997) M. E. A Reith, ed. Human Press, Towata N.J., and thereferences cited therein.

[0096] A typical combinatorial chemical library is a collection ofdiverse chemical compounds generated by either chemical synthesis orbiological synthesis, by combining a number of chemical “buildingblocks” such as reagents. For example, a linear combinatorial chemicallibrary such as a polypeptide library is formed by combining a set ofchemical building blocks (amino acids) in every possible way, or aselected way for a given compound length (i.e., the number of aminoacids in a polypeptide compound). Millions of chemical compounds can besynthesized through such combinatorial mixing of chemical buildingblocks.

[0097] Preparation and screening of combinatorial chemical libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (PCT PublicationNo. WO 91/19735), encoded peptides (PCT Publication WO 93/20242), randombio-oligomers (PCT Publication No. WO 92/00091), benzodiazepines (U.S.Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines anddipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913(1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.114:6568 (1992)), nonpeptidal peptidomimetics with a-D-glucosescaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218(1992)), analogous organic syntheses of small compound libraries (Chenet al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho etal., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell etal., J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see, Bergerand Kimmel, Guide to Molecular Cloning Techniques. Methods in Enzymologyvolume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook etal. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3,Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook);and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (e.g., current through 1999, e.g., atleast through supplement 37) (Ausubel)), peptide nucleic acid libraries(see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g.,Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) andPCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al.,Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, 5,288,514, and thelike). Diverse shuffled libraries of nucleic acids are optionallyprovided, e.g., using fast forced evolution techniques, e.g., as in U.S.Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252 and 5,837,458.

[0098] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, MD, etc.).

[0099] Control reactions which measure TMP or activity of a selected TMPmodulator which does not include the modulator are optional, as theassays can be performed in a uniform fashion. Such optional controlreactions are generally appropriate, however, and increase thereliability of the assay(s). Accordingly, in one embodiment, the methodsof the invention include a control reaction (or reactions). For each ofthe assay formats described, “no modulator” control reactions which donot include a modulator provide a background level of transporteractivity. “Control modulator” reactions which have a known activity onTMP in a particular assay can also be run.

[0100] In some assays, it is desirable to have positive controls toensure that the components of the assays are working properly. At leasttwo types of positive controls are appropriate. First, a known activatorof ion channels, ionophores, or ion transporters can be flowed intocontact with a membrane component comprising transport activities, andthe resulting affects on TMP activity monitored. Second, a knowninhibitor of ion channels or transporters can be added, and theresulting effects in TMP activity similarly detected. It will beappreciated that modulators can also be combined in assays with knownactivators or inhibitors to find modulators which inhibit activation orrepression of activity by the known activator or inhibitor.

[0101] TMP Assays

[0102] In accordance with the methods of the invention, the change inthe level of fluorescence of the composition is detected, where thechange in fluorescence is indicative of a change in transmembranepotential. Typically, the assay methods described herein are used todetect the effect of some stimulus on the functioning of a cellularsystem. Where one is seeking to determine the effect of some stimulus ona cell's transmembrane potential, e.g., through a change in ion flux,transport, membrane permeability, or the like, one need only expose thecell to that stimulus and examine the cell for the presence of apreviously absent fluorescent signal (or the absence of a previouslypresent fluorescent signal). Of particular interest are the effects ofchemical compounds, e.g., drug candidates, on cellular functioning, asdeterminable from TMP measurements.

[0103] For example, in one assay format, a dye is contacted to amembrane composition. In accordance with these methods, the membranecomposition is typically placed into a reaction vessel, such as amicrofluidic channel, and the level of fluorescence from the compositionis measured, optionally over a period of time. This can be used toprovide an initial or background level of fluorescence indicative of anexisting transmembrane potential for the cell population. The particularstimulus that is to be tested is then inflicted upon the cellpopulation. For example, a pharmaceutical candidate or test compound isadded to the cell population. Following this stimulus, the fluorescencelevel of the cells is again measured (typically over time) and comparedto the initial fluorescent level or the fluorescence level in a controlcell population (e.g., which is exposed to a control TMP modulator). Anychange in the level of fluorescence not attributable to dilution by thetest compound (as determined from an appropriate control) is thenattributable to the effect the test compound has on the cell'stransmembrane potential, or rate of TMP change in response todepolarization or hyperpolarization events.

[0104] As described in greater detail below, typically, these types ofreactions are carried out in an appropriate reaction receptacle thatallows measurement of fluorescence, in situ. As such, the receptacle istypically a transparent reaction vessel, such as a test tube, cuvette, areaction well in a multiwell plate, or a transparent conduit, e.g., acapillary, microchannel or tube. In particularly preferred aspects, theassay methods are carried out in the channel or channels of amicrofluidic device, as described in greater detail below.

[0105] The assay methods of the present invention are particularlyuseful in performing high-throughput (greater than 1,000 compounds/day)and even ultra-high throughput (e.g., greater than 10,000 compounds/day)screening of chemical libraries, e.g., in searching for pharmaceuticalleads. These experiments may be carried out in parallel by a providing alarge number of reaction mixtures (e.g., cell suspensions as describedherein) in separate receptacles, typically in a multiwell format, e.g.,96 well, 324 well or 1536 well plates. Different test compounds (librarymembers) are added to separate wells, and the effect of the compound onthe reaction mixture is ascertained, e.g., via the fluorescent signal.These parallelized assays are generally carried out using specializedequipment to enable simultaneous processing of large numbers of samples,i.e., fluid handling by robotic pipettor systems and fluorescentdetection by multiplexed fluorescent multi-well plate readers.

[0106] In an alternative aspect, the assays are carried out, at least inpart, in a serial format, where separate samples are screened one afteranother for an effect on a cellular system or other membranepreparation. In order to expand throughput, these individual serialprocessing units themselves may be multiplexed or parallelized. Inparticularly preferred aspects, the serial assays are performed within amicrofluidic device or system. Examples of these microfluidic devicesand systems are described in Published International Patent ApplicationNo. WO 98/00231, which is incorporated herein by reference in itsentirety for all purposes.

[0107] Assay Systems

[0108] The present invention provides assay systems for carrying out theassay methods of the present invention. Briefly and as noted above, suchsystems typically employ a reaction or assay receptacle in which thecompositions of the invention are disposed. Additional reagents may beadded, e.g., as potential or actual inhibitors or enhancers of thereaction of interest (involving TMP measurement). Typically, thereceptacle includes at least a portion that is transparent, so that afluorescent signal from the dye may be detected. Of course, in the caseof test tubes or wells, detection can be made through an opening in thereceptacle, e.g., the top opening of a well. A variety of receptaclesare useful in the present invention, including individual test tubes,cuvettes, wells in a multiwell plate, or capillary tubes.

[0109] Microfluidic and Integrated Systems for TMP Measurements and HighThroughput Detection of TMP Modulators

[0110] A variety of microscale systems which can be adapted to thepresent invention by incorporating transporter components, transmittercomponents, modulators and the like are available. Microfluidic deviceswhich can be adapted to the present invention by the addition of TMPassay components are described in various PCT applications and issuedU.S. Patents by the inventors and their coworkers, including U.S. Pat.Nos. 5,699,157 (J. Wallace Parce) issued Dec. 16, 1997, U.S. Pat. No.5,779,868 (J. Wallace Parce et al.) issued Jul. 14, 1998, U.S. Pat. No.5,800,690 (Calvin Y. H. Chow et al.) issued Sep. 1, 1998, and U.S. Pat.No. 5,842,787 (Anne R. Kopf-Sill et al.) issued Dec. 1, 1998; andpublished PCT applications, such as, WO 98/00231, WO 98/00705, WO98/00707, WO 98/02728, WO 98/05424, WO 98/22811, WO 98/45481, WO98/45929, WO 98/46438, and WO 98/49548.

[0111] For example, pioneering technology providing cell basedmicroscale assays are set forth in Parce et al. “High ThroughputScreening Assay Systems in Microscale Fluidic Devices” WO 98/00231 and,e.g., in PCT/US00/04522 filed Feb. 22, 2000, entitled MANIPULATION OFMICROPARTICLES IN MICROFLUIDIC SYSTEMS, by Mehta et al. Completeintegrated systems with fluid handling, signal detection, sample storageand sample accessing are available. For example, Parce et al. “HighThroughput Screening Assay Systems in Microscale Fluidic Devices” WO98/00231 provide pioneering technology for the integration ofmicrofluidics and sample selection and manipulation. Additionalreferences which provide additional details on manipulating cells inmicrofluidic systems, e.g., in cell focusing applications, cell sortingapplications high-throughput cell-based assays and the like, all ofwhich can be practiced in the context of the present invention, include:FOCUSING OF MICROPARTICLES IN MICROFLUIDIC SYSTEMS by Wada et al.,Application No: PCT/US00/13294, filed May 11, 2000, and HIGH THROUGPUTMETHODS, SYSTEMS AND APPARATUS FOR PERFORMING CELL BASED SCREENINGASSAYS by Wada et al., application no: PCT/US99/13918, filed Jun. 21,1999.

[0112] In general, cells, modulators, dyes, membrane components andother elements can be flowed in a microscale system by electrokinetic(including either electroosmotic or electrophoretic) techniques, orusing pressure-based flow mechanisms, or combinations thereof.

[0113] Cells in particular are desirably flowed using pressure-basedflow mechanisms. Pressure forces can be applied to microscale elementsto achieve fluid movement using any of a variety of techniques. Fluidflow (and flow of materials suspended or solubilized within the fluid,including cells or other particles) is optionally regulated by pressurebased mechanisms such as those based upon fluid displacement, e.g.,using a piston, pressure diaphragm, vacuum pump, probe or the like todisplace liquid and raise or lower the pressure at a site in themicrofluidic system. The pressure is optionally pneumatic, e.g., apressurized gas, or uses hydraulic forces, e.g., pressurized liquid, oralternatively, uses a positive displacement mechanism, i.e., a plungerfitted into a material reservoir, for forcing material through a channelor other conduit, or is a combination of such forces.

[0114] In other embodiments, a vacuum source is applied to a reservoiror well at one end of a channel to draw the suspension through thechannel. Pressure or vacuum sources are optionally supplied external tothe device or system, e.g., external vacuum or pressure pumps sealablyfitted to the inlet or outlet of the channel, or they are internal tothe device, e.g., microfabricated pumps integrated into the device andoperably linked to the channel. Examples of microfabricated pumps havebeen widely described in the art. See, e.g., published InternationalApplication No. WO 97/02357.

[0115] Hydrostatic, wicking and capillary forces can also be used toprovide pressure for fluid flow of materials such as cells. See, e.g.,“METHOD AND APPARATUS FOR CONTINUOUS LIQUID FLOW IN MICROSCALE CHANNELSUSING PRESSURE INJECTION, WICKING AND ELECTROKINETIC INJECTION,” byAlajoki et al., Attorney Docket Number 017646-007010, U.S. Ser. No.09/245,627, filed Feb. 5, 1999. In these methods, an adsorbent materialor branched capillary structure is placed in fluidic contact with aregion where pressure is applied, thereby causing fluid to move towardsthe adsorbent material or branched capillary structure.

[0116] Mechanisms for reducing adsorption of materials duringfluid-based flow are described in “PREVENTION OF SURFACE ADSORPTION INMICROCHANNELS BY APPLICATION OF ELECTRIC CURRENT DURING PRESSURE-INDUCEDFLOW” filed May 11, 1999 by Parce et al., Attorney Docket Number01-78-0, U.S. Ser. No. 09/310,027. In brief, adsorbtion of cells, TMPmodulators, dyes, potential modulators and other materials to channelwalls or other microscale components during pressure-based flow can bereduced by applying an electric field such as an alternating current tothe material during flow.

[0117] Mechanisms for focusing cells and other components into thecenter of microscale flow paths, which is useful in increasing assaythroughput by regularizing flow velocity is described in “FOCUSING OFMICROPARTICLES IN MICROFLUIDIC SYSTEMS” by H. Garrett Wada et al.Application No: PCT/US00/13294, filed May 11, 2000. In brief, cells arefocused into the center of a channel by forcing fluid flow from opposingside channels into the main channel comprising the cells, or by otherfluid manipulations. Diffusible materials such as the transmitters ofthe present invention are also optionally washed from cells as describedby Wada et al. during flow of the cells, i.e., by sequentially flowingbuffer into a channel in which cells are flowed and flowing the bufferback out of the channel.

[0118] In an alternate embodiment, microfluidic systems can beincorporated into centrifuge rotor devices, which are spun in acentrifuge. Fluids and particles travel through the device due togravitational and centripetal/centrifugal pressure forces.

[0119] One method of achieving transport or movement of dyes, TMPmodulators, and even cells (particularly dyes and modulators) throughmicrofluidic channels is by electrokinetic material transport.“Electrokinetic material transport systems,” as used herein, includesystems that transport and direct materials within a microchannel and/orchamber containing structure, through the application of electricalfields to the materials, thereby causing material movement through andamong the channel and/or chambers, i.e., cations will move toward anegative electrode, while anions will move toward a positive electrode.For example, movement of fluids toward or away from a cathode or anodecan cause movement of transmitters, cells, modulators, etc. suspendedwithin the fluid. Similarly, the transmitters, cells, modulators, etc.can be charged, in which case they will move toward an oppositelycharged electrode (indeed, in this case, it is possible to achieve fluidflow in one direction while achieving particle flow in the oppositedirection). In this embodiment, the fluid can be immobile or flowing andcan comprise a matrix as in electrophoresis.

[0120] In general, electrokinetic material transport and directionsystems also include those systems that rely upon the electrophoreticmobility of charged species within the electric field applied to thestructure. Such systems are more particularly referred to aselectrophoretic material transport systems. For electrophoreticapplications, the walls of interior channels of the electrokinetictransport system are optionally charged or uncharged. Typicalelectrokinetic transport systems are made of glass, charged polymers,and uncharged polymers. The interior channels are optionally coated witha material which alters the surface charge of the channel.

[0121] A variety of electrokinetic controllers and systems aredescribed, e.g., in Ramsey WO 96/04547, Parce et al. WO 98/46438 andDubrow et al., WO 98/49548, as well as a variety of other referencesnoted herein.

[0122] Use of electrokinetic transport to control material movement ininterconnected channel structures was described, e.g., in WO 96/04547and U.S. Pat. No. 5,858,195 to Ramsey. An exemplary controller isdescribed in U.S. Pat. No. 5,800,690. Modulating voltages areconcomitantly applied to the various reservoirs to affect a desiredfluid flow characteristic, e.g., continuous or discontinuous (e.g., aregularly pulsed field causing the sample to oscillate direction oftravel) flow of labeled components toward a waste reservoir.Particularly, modulation of the voltages applied at the variousreservoirs can move and direct fluid flow through the interconnectedchannel structure of the device.

[0123] Sources of Assay Components and Integration with MicrofluidicFormats

[0124] Sources of membrane containing components such as cells or cellfractions, sources of dyes and sources of potential modulators can befluidly coupled to the microchannels noted herein in any of a variety ofways. In particular, those systems comprising sources of materials setforth in Knapp et al. “Closed Loop Biochemical Analyzers” (WO 98/45481;PCT/US98/06723) and Parce et al. “High Throughput Screening AssaySystems in Microscale Fluidic Devices” WO 98/00231 and, e.g., inPCT/US00/04522 filed Feb. 22, 2000, entitled MANIPULATION OFMICROPARTICLES IN MICROFLUIDIC SYSTEMS, by Mehta et al. are applicable.

[0125] In these systems, a “pipettor channel” (a channel in whichcomponents can be moved from a source to a microscale element such as asecond channel or reservoir) is temporarily or permanently coupled to asource of material. The source can be internal or external to amicrofluidic device comprising the pipettor channel. Example sourcesinclude microwell plates, membranes or other solid substrates comprisinglyophilized components, wells or reservoirs in the body of themicroscale device itself and others.

[0126] For example, the source of a cell type, component, or modulatorreagent can be a microwell plate external to the body structure, having,e.g., at least one well with the selected cell type or reagent.Alternatively, a well disposed on the surface of the body structurecomprising the selected cell type, component, or reagent, a reservoirdisposed within the body structure comprising the selected cell type,component or reagent; a container external to the body structurecomprising at least one compartment comprising the selected particletype or reagent, or a solid phase structure comprising the selected celltype or reagent in lyophilized or otherwise dried form.

[0127] A loading channel region is optionally fluidly coupled to apipettor channel with a port external to the body structure, e.g., asdepicted in FIGS. 6, and 7. The loading channel can be coupled to anelectropipettor channel with a port external to the body structure, apressure-based pipettor channel with a port external to the bodystructure, a pipettor channel with a port internal to the bodystructure, an internal channel within the body structure fluidly coupledto a well on the surface of the body structure, an internal channelwithin the body structure fluidly coupled to a well within the bodystructure, or the like. Example configurations are depicted in thefigures herein.

[0128] As described more fully herein, the integrated microfluidicsystem of the invention can include a very wide variety of storageelements for storing reagents to be assessed. These include well plates,matrices, membranes and the like. The reagents are stored in liquids(e.g., in a well on a microtiter plate), or in lyophilized form (e.g.,dried on a membrane or in a porous matrix), and can be transported to anarray component of the microfluidic device using conventional robotics,or using an electropipettor or pressure pipettor channel fluidly coupledto a reaction or reagent channel of the microfluidic system.

[0129] In general, the test modulator compounds are separatelyintroduced into the assay systems described herein, or at leastintroduced in relatively manageable pools of modulator materials. Therelative level of a particular TMP function is then assessed in thepresence of the test compound, and this relative level of function isthen compared to a control system, which lacks an introduced testmodulator compound. Increases or decreases in relative cellular functionare indicative that the test compound is an enhancer or an inhibitor ofthe particular cellular function, respectively.

[0130] Detectors and Integrated Systems

[0131] Although the devices and systems specifically illustrated hereinare generally described in terms of the performance of a few or oneparticular operation, it will be readily appreciated from thisdisclosure that the flexibility of these systems permits easyintegration of additional operations into these devices. For example,the devices and systems described will optionally include structures,reagents and systems for performing virtually any number of operationsboth upstream and downstream from the operations specifically describedherein. Such upstream operations include sample handling and preparationoperations, e.g., cell separation, extraction, purification, culture,amplification, cellular activation, labeling reactions, dilution,aliquotting, and the like. Similarly, downstream operations may includesimilar operations, including, e.g., separation of sample components,labeling of components, assays and detection operations, electrokineticor pressure-based injection of components into contact with cells orother membrane preparations, or materials released from cells ormembrane preparations, or the like.

[0132] Upstream and downstream assay and detection operations include,without limitation, cell fluorescence assays, cell activity assays,probe interrogation assays, e.g., nucleic acid hybridization assaysutilizing individual probes, free or tethered within the channels orchambers of the device and/or probe arrays having large numbers ofdifferent, discretely positioned probes, receptor/ligand assays,immunoassays, and the like. Any of these elements can be fixed to arraymembers, or fixed, e.g., to channel walls, or the like.

[0133] Instrumentation for high throughput optical screening of cellassays is available. In addition to the many microfluidic systems notedherein, other automated approaches can also be practiced with the dyesand methods of the invention. For example, the FLIPR (FluorescenceImaging Plate Reader) was developed to perform quantitative opticalscreening for cell based kinetic assays (Schroder and Neagle (1996)“FLIPR: A New Instrument for Accurate, High Throughput OpticalScreening” Journal of Biomolecular Screening 1(2):75-80). This devicecan be adapted to the present invention, e.g., by using the dyes of theinvention in the indicated methods. For example, cationic DNA membranepermeable dyes can be used for potentiometric measurements. Similarly,by acquiring data on dye uptake prior to establishing equilibrium forboth cationic and anionic dyes, the system can be adapted to theinvention.

[0134] Instrumentation

[0135] In general in the present invention, materials such as cells anddyes are optionally monitored and/or detected so that an activity suchas TMP activity can be determined. Depending on the label signalmeasurements, decisions can be made regarding subsequent fluidicoperations, e.g., whether to assay a particular modulator in detail todetermine kinetic information.

[0136] The systems described herein generally include microfluidicdevices, as described above, in conjunction with additionalinstrumentation for controlling fluid transport, flow rate and directionwithin the devices, detection instrumentation for detecting or sensingresults of the operations performed by the system, processors, e.g.,computers, for instructing the controlling instrumentation in accordancewith preprogrammed instructions, receiving data from the detectioninstrumentation, and for analyzing, storing and interpreting the data,and providing the data and interpretations in a readily accessiblereporting format.

[0137] Controllers

[0138] A variety of controlling instrumentation is optionally utilizedin conjunction with the microfluidic devices described above, forcontrolling the transport and direction of fluids and/or materialswithin the devices of the present invention, e.g., by pressure-based orelectrokinetic control.

[0139] For example, in many cases, fluid transport and direction arecontrolled in whole or in part, using pressure based flow systems thatincorporate external or internal pressure sources to drive fluid flow.Internal sources include microfabricated pumps, e.g., diaphragm pumps,thermal pumps, Lamb wave pumps and the like that have been described inthe art. See, e.g., U.S. Pat. Nos. 5,271,724, 5,277,556, and 5,375,979and Published PCT Application Nos. WO 94/05414 and WO 97/02357. As notedabove, the systems described herein can also utilize electrokineticmaterial direction and transport systems.

[0140] Preferably, external pressure sources are used, and applied toports at channel termini. These applied pressures, or vacuums, generatepressure differentials across the lengths of channels to drive fluidflow through them. In the interconnected channel networks describedherein, differential flow rates on volumes are optionally accomplishedby applying different pressures or vacuums at multiple ports, orpreferably, by applying a single vacuum at a common waste port andconfiguring the various channels with appropriate resistance to yielddesired flow rates. Example systems are described in U.S. Ser. No.09/238,467 filed Jan. 28, 1999.

[0141] Typically, the controller systems are appropriately configured toreceive or interface with a microfluidic device or system element asdescribed herein. For example, the controller and/or detector,optionally includes a stage upon which the device of the invention ismounted to facilitate appropriate interfacing between the controllerand/or detector and the device. Typically, the stage includes anappropriate mounting/alignment structural element, such as a nestingwell, alignment pins and/or holes, asymmetric edge structures (tofacilitate proper device alignment), and the like. Many suchconfigurations are described in the references cited herein.

[0142] The controlling instrumentation discussed above is also used toprovide for electrokinetic injection or withdrawal of materialdownstream of the region of interest to control an upstream flow rate.The same instrumentation and techniques described above are alsoutilized to inject a fluid into a downstream port to function as a flowcontrol element.

[0143] Detector

[0144] The devices herein optionally include signal detectors, e.g.,which detect fluorescence, phosphorescence, radioactivity, pH, charge,absorbance, luminescence, temperature, magnetism or the like.Fluorescent detection is especially preferred and generally used fordetection of voltage sensitive compounds (however, as noted, upstreamand downstream operations can be performed on cells, dyes, modulators orthe like, which can involve other detection methods).

[0145] The detector(s) optionally monitors one or a plurality of signalsfrom downstream of an assay mixing point in which dye and a cell orother membrane containing component with a potential modulator aremixed. For example, the detector can monitor a plurality of opticalsignals which correspond to “real time” assay results.

[0146] Example detectors include photo multiplier tubes,spectrophotometers, a CCD array, a scanning detector, a microscope, agalvo-scann or the like. Cells, dyes or other components which emit adetectable signal can be flowed past the detector, or, alternatively,the detector can move relative to the array to determine cell position(or, the detector can simultaneously monitor a number of spatialpositions corresponding to channel regions, e.g., as in a CCD array).

[0147] The detector can include or be operably linked to a computer,e.g., which has software for converting detector signal information intoassay result information (e.g., kinetic data of modulator activity), orthe like.

[0148] Signals are optionally calibrated, e.g., by calibrating themicrofluidic system by monitoring a signal from a known source.

[0149] A microfluidic system can also employ multiple differentdetection systems for monitoring the output of the system. Detectionsystems of the present invention are used to detect and monitor thematerials in a particular channel region (or other reaction detectionregion). Once detected, the flow rate and velocity of cells in thechannels is also optionally measured and controlled as described above.As described in PCT/US98/11969, and No. 60/142,984, correction ofkinetic information based upon flow velocity and other factors can beused to provide accurate kinetic information in flowing systems.

[0150] Examples of detection systems include optical sensors,temperature sensors, pressure sensors, pH sensors, conductivity sensors,and the like. Each of these types of sensors is readily incorporatedinto the microfluidic systems described herein. In these systems, suchdetectors are placed either within or adjacent to the microfluidicdevice or one or more channels, chambers or conduits of the device, suchthat the detector is within sensory communication with the device,channel, or chamber. The phrase “within sensory communication” of aparticular region or element, as used herein, generally refers to theplacement of the detector in a position such that the detector iscapable of detecting the property of the microfluidic device, a portionof the microfluidic device, or the contents of a portion of themicrofluidic device, for which that detector was intended. For example,a pH sensor placed in sensory communication with a microscale channel iscapable of determining the pH of a fluid disposed in that channel.Similarly, a temperature sensor placed in sensory communication with thebody of a microfluidic device is capable of determining the temperatureof the device itself.

[0151] Particularly preferred detection systems include opticaldetection systems for detecting an optical property of a material withinthe channels and/or chambers of the microfluidic devices that areincorporated into the microfluidic systems described herein. Suchoptical detection systems are typically placed adjacent to a microscalechannel of a microfluidic device, and are in sensory communication withthe channel via an optical detection window that is disposed across thechannel or chamber of the device. Optical detection systems includesystems that are capable of measuring the light emitted from materialwithin the channel, the transmissivity or absorbance of the material, aswell as the materials spectral characteristics. In preferred aspects,the detector measures an amount of light emitted from the material, suchas a fluorescent or chemiluminescent material. As such, the detectionsystem will typically include collection optics for gathering a lightbased signal transmitted through the detection window, and transmittingthat signal to an appropriate light detector. Microscope objectives ofvarying power, field diameter, and focal length are readily utilized asat least a portion of this optical train. The light detectors areoptionally spectrophotometers, photodiodes, avalanche photodiodes,photomultiplier tubes, diode arrays, or in some cases, imaging systems,such as charged coupled devices (CCDs) and the like. The detectionsystem is typically coupled to a computer (described in greater detailbelow), via an analog to digital or digital to analog converter, fortransmitting detected light data to the computer for analysis, storageand data manipulation.

[0152] In the case of fluorescent materials such as labeled cells, thedetector typically includes a light source which produces light at anappropriate wavelength for activating the fluorescent material, as wellas optics for directing the light source through the detection window tothe material contained in the channel or chamber. The light source canbe any number of light sources that provides an appropriate wavelength,including lasers, laser diodes and LEDs. Other light sources are used inother detection systems. For example, broad band light sources aretypically used in light scattering/transmissivity detection schemes, andthe like. Typically, light selection parameters are well known to thoseof skill in the art.

[0153] The detector can exist as a separate unit, but can also beintegrated with the controller system, into a single instrument.Integration of these functions into a single unit facilitates connectionof these instruments with the computer (described below), by permittingthe use of few or a single communication port(s) for transmittinginformation between the controller, the detector and the computer.

[0154] Computer

[0155] As noted above, either or both of the controller system and/orthe detection system are coupled to an appropriately programmedprocessor or computer which functions to instruct the operation of theseinstruments in accordance with preprogrammed or user input instructions,receive data and information from these instruments, and interpret,manipulate and report this information to the user. As such, thecomputer is typically appropriately coupled to one or both of theseinstruments (e.g., including an analog to digital or digital to analogconverter as needed).

[0156] The computer typically includes appropriate software forreceiving user instructions, either in the form of user input into a setparameter fields, e.g., in a GUI, or in the form of preprogrammedinstructions, e.g., preprogrammed for a variety of different specificoperations. The software then converts these instructions to appropriatelanguage for instructing the operation of the fluid direction andtransport controller to carry out the desired operation. The computerthen receives the data from the one or more sensors/detectors includedwithin the system, and interprets the data, either provides it in a userunderstood format, or uses that data to initiate further controllerinstructions, in accordance with the programming, e.g., such as inmonitoring and control of flow rates, temperatures, applied voltages,and the like.

[0157] In the present invention, the computer typically includessoftware for the monitoring of materials in the channels. Additionally,the software is optionally used to control electrokinetic or pressuremodulated injection or withdrawal of material. The injection orwithdrawal is used to modulate the flow rate as described above, to mixcomponents, and the like.

[0158] Example System

[0159]FIG. 6, panels A, B and C and FIG. 7 provide additional detailsregarding an example integrated system of the invention. As shown, bodystructure 102 has main channel 104 fabricated therein. Cells or othermembrane containing components are flowed, e.g., from reservoir 114,e.g., by applying a vacuum at vacuum source 116 (and/or at any of thereservoirs or wells noted below) through main channel 104. Cells orother membrane containing components, or dye(s), or a potentialmodulator or a different material such as a buffer or label can beflowed from wells 110 or 112 and into main channel 104. Cells, or dye,or a potential modulator or any additional material can be flowed fromwells 106 or 108, or materials can be flowed into these wells, e.g.,when they are used as waste wells, or when they are coupled to a vacuumsource. Flow from wells 114, 112, 110, 106, or 108 can be performed bymodulating fluid pressure, or by electrokinetic approaches as described.Instead of the arrangement of channels depicted in FIGS. 6 and 7, anarrangement such as the 3B5 microfluidic processor, as depicted in FIG.5 can be substituted. A variety of other appropriate microfluidicprocessor configurations are set forth in the references noted herein.

[0160] With respect to FIG. 5, device 501 comprises cell well 503, sidewell 505 (e.g., a sample well) and side well 507 (e.g., a dye well) andwaste well 509. In brief, cells are flowed from well 503, through cellfeed channel 511, and through main channel 513 to vacuum at waste well509. Sample is flowed from side well 505 through sample feed channel 515and into main channel 513. Dye is flowed from side well 507 through dyefeed channel 517 and into main channel 513. As noted, flow is directedvia application of vacuum at waste well 509. In this particular device,the approximate channel dimensions are 25 μm deep and 100 μm wide.Channel 511 is approximately 5.2 mm in length. Channel 515 isapproximately 13.4 mm in length. Channel 517 is approximately 13.4 mm inlength. Channel 513 is approximately 32.3 mm in length.

[0161] Cells, membrane preparations, dyes, potential modulators or othermaterials can be flowed from the enumerated wells, or can be flowed froma source external to body 102. As depicted, the integrated system caninclude pipettor channel 120, e.g., protruding from body 102, foraccessing an outside source of reagents. For example, as furtherdepicted in FIG. 7, pipettor channel 120 can access microwell plate 308which includes cells, dyes, activity modulators, controls, or the like,in the wells of the plate. For example, a library of potential inhibitorcompounds can be stored in the wells of plate 308 for easy access by thesystem. TMP change inhibitors, activators or other reagents relevant tothe assays can be flowed into channel 104 through pipettor channel 120.Detector 306 is in sensory communication with channel 104, detectingsignals resulting, e.g., from the interaction of a dye with a cell orother membrane preparation, as described above. Detector 306 is operablylinked to Computer 304, which digitizes, stores and manipulates signalinformation detected by detector 306. Voltage/pressure controller 302controls voltage, pressure, or both, e.g., at the wells of the system,or at vacuum couplings fluidly coupled to channel 104 (or the otherchannels noted above). Optionally, as depicted, computer 304 controlsvoltage/pressure controller 302. In one set of embodiments, computer 304uses signal information to select further reaction parameters. Forexample, upon detecting inhibition or activation by a potentialmodulator in a well from plate 308, the computer optionally directswithdrawal of additional aliquots of the potential modulator throughpipettor channel 120, e.g., to deliver different concentrations of thepotential modulator to the assay, e.g., to determine kinetic data (suchas a dose-response curve) for the potential modulator.

[0162] Assay Kits

[0163] The present invention also provides kits for carrying out theassay methods described herein. In particular, these kits typicallyinclude the compositions described herein, as well as additionalcomponents to facilitate the performance of the assay methods by aninvestigator. In particular, the kits typically comprise the voltagesensing composition of the invention.

[0164] The kit also optionally includes a receptacle in which the assayreaction is carried out. As noted herein, the reaction receptacle isoptionally a reaction vessel, i.e., a test tube or well in a multiwellplate, or a channel or chamber region within a microfluidic device. Thereaction receptacle is also typically transparent, at least in part, inorder to detect the fluorescent signals from the reaction mixture.

[0165] The elements of the kits of the present invention are typicallypackaged together in a single package or set of related packages. Thepackage optionally includes other reagents used in the assay, e.g.,buffers, standard reagents, and the like, as well as writteninstructions for carrying out the assay in accordance with the methodsdescribed herein. In the case of prepackaged reagents, the kitsoptionally include pre-measured or pre-dosed reagents that are ready toincorporate into the assay methods without measurement, e.g.,pre-measured fluid aliquots, or pre-weighed or pre-measured solidreagents that may be easily reconstituted by the end-user of the kit.

[0166] Generally, the microfluidic devices described herein areoptionally packaged to include reagents for performing the device'spreferred function. For example, the kits can include any ofmicrofluidic devices described along with assay components, reagents,sample materials, control materials, or the like. Such kits alsotypically include appropriate instructions for using the devices andreagents, and in cases where reagents are not predisposed in the devicesthemselves, with appropriate instructions for introducing the reagentsinto the channels and/or chambers of the device. In this latter case,these kits optionally include special ancillary devices for introducingmaterials into the microfluidic systems, e.g., appropriately configuredsyringes/pumps, or the like (in one preferred embodiment, the deviceitself comprises a pipettor element, such as an electropipettor forintroducing material into channels and chambers within the device). Inthe former case, such kits typically include a microfluidic device withnecessary reagents predisposed in the channels/chambers of the device.Generally, such reagents are provided in a stabilized form, so as toprevent degradation or other loss during prolonged storage, e.g., fromleakage. A number of stabilizing processes are widely used for reagentsthat are to be stored, such as the inclusion of chemical stabilizers(i.e., enzymatic inhibitors, microcides/bacteriostats, anticoagulants),the physical stabilization of the material, e.g., through immobilizationon a solid support, entrapment in a matrix (i.e., a gel),lyophilization, or the like.

[0167] Kits also optionally include packaging materials or containersfor holding microfluidic device, system or reagent elements,instructional materials for practicing any of the methods noted herein,or the like.

EXAMPLE Development of Microfluidic Processor-Based TransmembranePotential Assays

[0168] Measurement of transmembrane potential is useful for a broadclass of cell based microfluidic assays. A widely used type oftransmembrane potential assay uses voltage-sensitive dyes which generatea fluorescent signal due to voltage-dependent, dye redistributionbetween the inside and outside of cells. The feasibility of using suchNernstian dyes to measure transmembrane potential in microfluidicprocessors was investigated.

[0169] Typically, the use of Nemstian, voltage-sensitive dyes such asDiBAC₄(3) involves the measurement of the equilibrium distribution ofdye as a function of transmembrane potential. Alternatively, it wasfound that the addition of DiBAC₄(3) to cell suspensions resulted in avoltage-dependent time course of dye uptake (FIG. 1).

[0170] As shown in FIG. 1, the time course of dye uptake depends ontransmembrane potential. THP-1 cells (3×10⁵ cells/ml) were suspended ineither high sodium, Hepes-Hanks balanced salt solution (143 mM Na⁺, 2 mMK⁺) (line A) or high potassium, Hepes-Hanks balanced salt solution (15mM Na⁺, 130 mM K⁺) containing 15% Optiprep and 200 nM DiBAC₄(3) (lineB). The cells in high sodium and high potassium buffers are expected tohave transmembrane potentials of about −40 mV and 0 mV respectively. Thechange in DiBAC₄(3) fluorescence (475 nm excitation, 520 nm emission)after suspension of the cells was measured spectrophotometrically at 30°C.

[0171] Surprisingly, it was found that the DNA stain SYTO62, acyclic-substituted unsymmetrical cyanine dye (see, e.g., U.S. Pat. No.5,436,134), acts as a voltage sensitive dye.

[0172] In particular, as shown in FIG. 2, the time course of SYTO 62uptake depends on transmembrane potential. THP-1 cells were suspended inhigh sodium (line A) or high potassium (line B) buffers containing 1 μMSYTO 62 as for the experiments depicted in FIG. 1. The change in SYTO62fluorescence (475 nm excitation, 670 nm emission) after cell suspensionwas measured spectrophotometrically at 30° C. Membrane permeable,cationic, ribonucleic acid stains such as SYTO62 thus represent a newclass of Nemstian voltage dyes.

[0173] Changes in the cell transmembrane potential were detected inmicrofluidic processor by the mixing of cells and the dyes DiBAC₄(3) andSYTO62. As shown in FIG. 3, transmembrane potential assays can be run ina microfluidic processor. Dye uptake by THP-1 cells was measured on aCaliper® 3B5 microfluidic processor (FIG. 5). One of the long channelsof the processor contained THP-1 cells (3×10⁶ cells/ml) suspended inhigh sodium buffer as above, while the other long channel contained 400nM DiBAC₄(3) and 10 μM SYTO62 in high sodium buffer. Transmembranepotential was set to resting potential, hyperpolarization ordepolarization by placing either high sodium, 50 μM UTP in high sodiumor high potassium buffer in the short arm. Resting, hyperpolarized anddepolarized potentials are estimated to be approximately −40, −100 and 0mV respectively. The microfluidic processor was maintained at roomtemperature (˜23° C.). DiBAC₄(3) and SYTO62 fluorescence was read afterthe cells had traveled approximately 90 seconds from the junction (ΔP˜1″H₂O). Panel A depicts the average fluorescence ratio (DiBAC₄(3)/SYTO62)for runs of ˜80 cells each. Error bars are standard error values. Asshown in panel B, Histograms show the expected voltage dependent changesin the fluorescence intensities of individual cells as a function oftransmembrane potential. Line A shows resting cells in high sodiumbuffer, line B shows depolarized cells in UTP/high potassium buffer andline C shows hyperpolarized cells in UTP/high sodium buffer.

[0174] The utility of the microfluidic processors-based assay wasdemonstrated by generating a Palytoxin dose-response curve as depictedin FIG. 4. Dye uptake by K562 cells (2×10⁶ cells/ml) was measured on a3B5 microfluidic processor as in FIG. 3, except that readings were made˜70 seconds after the junction. About 100 cells per condition were usedto calculate average values and standard errors. Solutions of varyingPalytoxin concentrations were placed in the short arm to yield theindicated Palytoxin concentrations after mixing with cells. Palytoxinopens a large Na⁺ current leading to a significant membranedepolarization.

[0175] The ability to measure transmembrane potential in microfluidicprocessors was demonstrated. By detecting the kinetics of dye uptakerather than the equilibrium dye distribution, the assay could be runquickly in microfluidic processors with high sensitivity. Membranepermeable, cationic ribonucleic acid stains such as SYTO62 were found tobe a new class of voltage sensitive dyes. The simultaneous use ofanionic and cationic dyes allows ratiometric measurement oftransmembrane potential leading to higher sensitivity and increaseddynamic range.

EXAMPLE Assays

[0176] The following Example illustrates the invention, but is notintended to limit the invention in any way. One of skill will be awareof a variety of substitutions that can be made in the following whileachieving substantially similar results.

[0177] The microfluidic technology used in the LabChip® system for thisexample utilizes microchannels etched into quartz chips coupled with asampling micropipette that can access samples from microtitre plates.The LabChip® system accesses liquid samples sequentially from amicroplate, mixes the samples with cells flowing through a microchanneland reads out cellular responses using fluorescence detection. Thesystem was applied to the measurement of changes in transmembranepotential. Membrane potential assays were demonstrated using membranepotential sensitive dyes to measure the hyperpolarization in THP-1 cellscaused by the opening of calcium-sensitive potassium channels afteraddition of UTP and the depolarization caused by blocking potassiumchannels by the addition of quinidine sulfate.

[0178] Lab-on-a-Chip System

[0179] As show in FIG. 8, panel A and panel B, the lab-on-a-chip systemwhich was used consisted of quartz chip 801 containing microchannels 803and 805 connected to SipperM capillary 807, robot 809 to move microplate811, light source 813, optics 815 and fluorescence reader 817, vacuumpump 816, and a computer to control system functions and record data.Light from light source 813 (e.g., mercury arc lamp (470 nm) or an argonion laser) was focused onto microchip 801 with through optics 815comprising an objective (10×, 0.3 NA air). Fluorescence was collectedwith the objective, split into two channels (525 nm and 700 nm) anddetected with fluorescence reader 817 which comprises photodiodes.Fluorescence readings were acquired at 20-90 Hz. The three axis robot809 was used to position microplate 811 so that chip 801 could samplemicroplate 811, whether in a 96 or 384 well configuration.

[0180] A schematic of chip 801 with 90×20 μm channels is shown in FIG.8, panel B. Flowing cells from cell well 819 were mixed with samplesintroduced through the Sipper™ capillary 807 (20 μm ID). The flow ratein the detection channel was varied from 2 to 10 nl/s by varying thevacuum applied to waste well 821. Agonists or dyes were added from sidewell 823. The incubation time in detection channel 803 was variedbetween 10 and 75 seconds by varying the applied pressure and thelocation of detector elements (e.g., optics 815) relative to chip 801.

[0181] Membrane Potential Assay

[0182] As shown in FIG. 9, The uptake of permeant fluorophores was asensitive indicator of membrane potential.

[0183] As shown in panel A, the potential sensitive fluorescence signalwas derived not from the slow re-equilibration of dyes that werepre-loaded into cells but rather from the rate of uptake of charged,membrane-permeable dyes. Using both an anionic dye, DiBAC₄(3), and acationic dye, Syto 62, yielded a ratiometric measurement with increasedsensitivity. Cells were mixed on chip with test samples and dyes storedon the chip. After a short incubation in the detection channel, thefluorescence of individual cells was detected.

[0184] As shown in panel B, peaks in green (DiBAC) and red (Syto 62)fluorescence corresponded to THP-1 cells mixed with either buffer, UTPor quinidine sulfate flowing past the detector following a 75 second dyeincubation.

[0185] As shown in panels C and D, the rate of uptake of DiBAC₄(3) (C)and Syto 62 (D) was determined from the average fluorescence peak heightas a function of the incubation time for resting cells (◯), cellshyperpolarized with UTP (▪) and cells depolarized with quinidine sulfate(▴). Compared to resting cells, the rate of increase of DiBAC₄(3) peakheights was 48±11% (mean±se, n=5) higher for depolarized cells and 43±5%lower for hyperpolarized cells. Similarly, the rate of increase of theSyto 62 peaks was 7±2% lower for depolarized cells and 37±3% higher forhyperpolarized cells.

[0186] Depolarization and Hyperpolarization

[0187] As shown in FIG. 10, depolarization and hyperpolarization weremeasured with the highly sensitive chip based assay.

[0188] As shown in panel A, a calibration curve relating the ratio ofDiBAC₄(3)/Syto 62 fluorescence (n=20 to 130 cells) to membrane potentialwas constructed by increasing the K+ conductance of the THP-1 cells andvarying the extracellular K+ concentration. The exponential curve fitindicated a doubling of the ratio for every 33 mV change in membranepotential. The average ratio for resting THP-1 cells in Hank's balancedsalt solution was 0.13 (n=15) yielding a resting membrane potential of−51 mV.

[0189] As shown in panel B, varying concentrations of UTP were sipped tohyperpolarize THP-1 cells. The dose dependent response of theDiBAC₄(3)/Syto 62 ratio (average±SD, n=6) to UTP is shown with an EC₅₀of 0.1 μM. As shown in panel C, the change in the average DiBAC₄(3)/Syto62 ratio (average±SD, n=3) due to quinidine sulfate dose dependentdepolarization is shown. The average coefficient of variance was 10% forreplicate measurements of 30 to 80 cells each. From the calibrationcurve, this corresponds to a 5 mV error in the estimation of themembrane potential.

[0190] Use of Primary Cells

[0191] As shown in FIG. 11, low cell consumption and high data qualityallowed the use of primary cells. Resting or mitogen activatedperipheral blood T lymphocytes (90% CD3+) were flowed through the chipand mixed with ion channel modulators accessed through the Sipper™capillary. Average DiBAC₄(3)/Syto 62 ratios±SE (n=2-6) are shown.Resting cells show a significant increase in DiBAC₄(3)/Syto 62 ratio (*,p<0.05) corresponding to a depolarization in the presence of 36 μMmargatoxin (BK_(Ca) blocker), but not 30 μM clotrimazole (IK_(Ca)blocker), 200 nM apamin (SK_(Ca) blocker) or 15 μM ionomycin (IK_(Ca)activator). In contrast, activated cells in the presence of ionomycinhad a lower ratio compared to resting T lymphocytes (*, p<0.05)corresponding to hyperpolarization. The ratio increases significantly inthe presence of 30 μM clotrimazole (**, p<0.05).

[0192] Thus, cell based assays of calcium flux and membrane potentialwere run in the lab-on-a-chip system. A novel approach for measurementof membrane potential was used to detect hyperpolarization anddepolarization with high sensitivity. The microfluidic format reducedreagent, sample and cell consumption. As few as 50-200 cells and aslittle as 10 nl of sample were used per test. The ability to use primarycells enabled by the low cell consumption was demonstrated by assayingion channel activity in human lymphocytes.

[0193]FIG. 12 shows a calibration curve relating the ratio ofDiBAC4(3)/Syto 62 fluorescence to membrane potential which wasconstructed for RBL-2H3 cells. RBL2H3 cells (ATCC CRL-2256) were grownin minimum essential Eagle medium with 2 mM L-glutamine and Earle's BSSadjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essentialamino acids, and 1.0 mM sodium pyruvate, 15% heat-inactivated fetalbovine serum and 100 u/ml penicillin, 100 μg/ml streptomycin at 37 μC in5% CO2. Cells were suspended by trypsinization and placed on chip inHBSS buffer containing 18% Optiprep. Membrane potential was modulated bytaking advantage of the large potassium conductance and varying theextracellular concentration of KCl from 2 to 66 mM. Membrane potentialswere calculated using the Goldman-Hodgkin-Katz equation. FIG. 12 showsthe calibration curve. The data was fit by an exponential curve(ratio=1.2×exp[0.019xpsi(mV)]) indicating a doubling of the ratio forevery 30 mV change in membrane potential. The average ratio for restingRBL-2H3 cells in Hank's balanced salt solution, was 0.26 correspondingto a resting membrane potential of −80 mV.

[0194] The discussion above is generally applicable to the aspects andembodiments of the invention described in the claims.

[0195] Moreover, modifications can be made to the method and apparatusdescribed herein without departing from the spirit and scope of theinvention as claimed, and the invention can be put to a number ofdifferent uses including the following:

[0196] The use of a microfluidic system for performing the TMP assaysset forth herein.

[0197] The use of a microfluidic system as described herein, wherein abiochemical system flows through one of said channels substantiallycontinuously, providing for, e.g., sequential testing of a plurality ofTMP modulatory compounds.

[0198] The use of pressure-based or electrokinetic injection in amicrofluidic device as described herein to modulate or achieve flow ofcells, membrane preparations, dyes, or other assay components inchannels of a microscale device.

[0199] The optional use of a combination of adsorbent materials,electrokinetic injection and pressure based flow elements in amicrofluidic device as described herein to modulate or achieve flow ofmaterials e.g., in the channels of the device.

[0200] An assay utilizing a use of any one of the microfluidic systemsor substrates described herein.

[0201] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. For example, all the techniques and apparatusdescribed above may be used in various combinations. All publications,patent applications, patents and other documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were individually so denoted.

What is claimed is:
 1. A composition, comprising: a first componentcomprising a membrane; a cationic membrane permeable dye; and, ananionic membrane permeable redistributing dye.
 2. The composition ofclaim 1, the cationic membrane permeable dye comprising a cationicnucleic acid staining dye.
 3. The composition of claim 1, wherein thefirst component comprises one or more of: a cell, a mitochondria, achloroplast, a cell vesicle, and an artificial membrane.
 4. Thecomposition of claim 3, wherein the cell comprises one or more of: anintact cell, an animal cell, a plant cell, a fungal cell, a bacterialcell, a mammalian cell, a primate cell, a rodent cell, a canine cell, afeline cell, and a livestock cell, a cultured cell, a THP-1 cell, a COScell, a CHO cell, a HEK cell, a HeLA cell, an NIH 3T3 cell, a primarycell, an endoderm cell, an ectoderm cell, a mesoderm cell, a cellderived from a differentiated tissue, a cell derived from anundifferentiated tissue, a blood cell, a peripheral blood cell, a nervecell, a muscle cell, a skin cell and a bone cell.
 5. The composition ofclaim 1, wherein the cationic membrane permeable dye comprises one ormore of: a Blue-fluorescent SYTO dye, a Green-fluorescent SYTO Dye, anOrange-fluorescent SYTO dye, a Red-fluorescent SYTO dye, SYTO 62, Pur-1,thiazol, aryl, 2DS-7J1, Hoechst 33258, Hoechst 33342 and hexidiumiodide; or wherein the anionic membrane permeable redistributing dyecomprises one or more of: an anionic bis-isoxazolone oxonol dye, abis-oxonol dye, Oxonol V, Oxonol VI, DiBAC₄(3) DiBAC₄(5), and DiBAC₂(3).6. A container or microfluidic processor comprising the composition ofclaim
 1. 7. A method of generating a signal output which is sensitive tomembrane potential, the method comprising: providing a first componentcomprising one or more membranes; adding a cationic membrane permeablenucleic acid staining dye to the first component; and, monitoring afirst signal output from the cationic membrane permeable nucleic acidstaining dye, wherein the first signal output is correlated with thetransmembrane potential across the one or more membranes.
 8. The methodof claim 7, the method comprising monitoring a change in transmembranepotential, the monitoring the first signal output comprising monitoringa change in the first signal output over time and the correlating thefirst signal output with the transmembrane potential across the one ormore membrane comprising determining the rate of change of the firstsignal over time.
 9. The method of claim 7, the providing stepcomprising flowing the first component through a first microfluidicchannel, which microfluidic channel intersects a second microfluidicchannel, wherein the cationic membrane permeable nucleic acid stainingdye is flowed through the first or second microchannel and into contactwith the at least one membrane.
 10. The method of claim 9, the methodcomprising flowing an anionic membrane permeable redistributing dye or aneutral dye through the first or second channel and into contact withthe at least one membrane.
 11. The method of claim 7, further comprisingexposing the one or more membranes to one or more transmembranepotential modulatory compositions and monitoring an effect of the one ormore transmembrane potential modulatory compositions on the first signaloutput, thereby monitoring an effect of the one or more transmembranepotential modulatory compositions on the transmembrane potential. 12.The method of claim 11, the one or more transmembrane potentialmodulatory compositions comprising one or more of: a hyperpolarizationbuffer, a depolarization buffer, and a compound which alters transportof an ion across the cell membrane.
 13. A method of producing a signalwhich is dependent on transmembrane potential, the method comprising:flowing a first mixture comprising one or more membranes and one or morevoltage sensitive dyes through a first channel region; and, monitoringat least a first signal output from at least one of the voltagesensitive dyes, thereby producing a signal which is dependent on thetransmembrane potential across the one or more membranes.
 14. The methodof claim 13, wherein the voltage sensitive dyes comprise one or moremembrane permeable redistributing dyes, which one or more membranepermeable dyes comprise one or more ionic dye and wherein the one ormore membrane permeable dyes are flowed from a source to the firstchannel region and into contact with the one or more membranes andwherein flow of the membrane permeable labels across the membrane isdetected by monitoring the one or more signal outputs from the membranepermeable labels before an equilibrium distribution is reached.
 15. Themethod of claim 13 or 14, wherein the first mixture comprises one ormore of: a cationic dye, a cationic membrane permeable nucleic acidstaining dye, an anionic dye and a neutral dye.
 16. The method of claim15, wherein the one or more voltage sensitive dyes comprises an anionicdye, a cationic dye, or a cationic membrane permeable nucleic acidstaining dye and one or more of: an anionic dye, Oxonol V, Oxonol VI,DiBAC₄(3), DiBAC₄(5), DiBAC₂(3), WW781, RGA-30, a cationic dye, anindo-carbocyanine dye with a short alkyl tail, a thio-carbocyanine dyewith a short alkyl tail, an oxacarbocyanine dye with a short alkyl tail,an amino napthyletheny pyridinium dye, a dialkyl amino phenylpolyenylpyridinium dye, a cationic membrane permeable nucleic acid staining dye,a SYTO dye, SYTO 62 and a neutral dye.
 17. The method of claim 13,wherein the first mixture is provided to the first channel by flowing afirst component comprising one or more membrane from a source to a firstchannel region and flowing a labeling composition comprising the one ormore voltage sensitive dyes into contact with the membrane.
 18. Themethod of claim 13, further comprising: hyperpolarizing or depolarizingthe membrane, or changing a permeability property of the membrane, andmonitoring flow of the at least one voltage sensitive dye across themembrane by monitoring the first signal output, thereby measuringchanges in the transmembrane potential.
 19. The method of claim 13,further comprising flowing at least a second mixture comprising one ormore second voltage sensitive dyes into contact with the membrane andmonitoring flow of the one or more second voltage sensitive dyes acrossthe membrane by monitoring at least a second signal output from thesecond voltage sensitive dyes.
 20. The method of claim 7, 13 or 19,comprising monitoring the first or second signal outputs over a selectedperiod of time (t), which period is less than about 100 seconds.
 21. Themethod of claim 20, wherein t is between about 0.1 and about 80 seconds.22. The method of claim 7, 13 or 19, wherein the first or second signaloutput is monitored at one or more time points, which one or more timepoints are before equilibration of the first voltage sensitive dyes, theat least second voltage sensitive dyes or the cationic membranepermeable nucleic acid staining dye across the one or more membranes.23. The method of claim 13, wherein the voltage sensitive dye is acationic membrane permeable nucleic acid staining dye.
 24. The method ofclaim 7, 13 or 23, wherein a rate of dye translocation across themembrane depends on the transmembrane potential across the membrane. 25.The method of claim 7 or 23, wherein the cationic membrane permeablenucleic acid staining dye is a cyanine dye, or a cyclic-substitutedunsymmetrical cyanine dye.
 26. The method of claim 7 or 23, wherein thecationic membrane permeable nucleic acid staining dye is a dye selectedfrom: a Blue-fluorescent SYTO dye, a Green-fluorescent SYTO Dye, anOrange-fluorescent SYTO dye, a Red-fluorescent SYTO dye, Pur-1, thiazol,aryl, 2DS-7J1, Hoechst 33258, Hoechst 33342 and hexidium iodide.
 27. Themethod of claim 7 or 23, wherein the dye is the Red-fluorescent dye SYTO62.
 28. The method of claim 7 or 23, the method comprising adding ananionic membrane permeable redistributing dye to the first component orto the first mixture and measuring a second signal output from theanionic membrane permeable redistributing dye, thereby providing afurther indication of changes in the transmembrane potential.
 29. Themethod of claim 28, further comprising determining a ratio of the firstand second signal.
 30. The method of claim 28, wherein the anionicmembrane permeable redistributing dye comprises one or more of: ananionic bis-isoxazolone oxonol dye, a bis-oxonol dye, Oxonol V, OxonolVI, DiBAC₄(3), DiBAC₄(5), and DiBAC₂(3).
 31. The method of claim 28,wherein the cationic membrane permeable nucleic acid staining dye isSYTO 62, at a concentration of between about 0.01 and about 50 μM andthe anionic dye is DiBAC₄(3), at a concentration of between about 0.01and about 50 μM.
 32. The method of claim 7 or 13, wherein the firstmembrane is a component of an intact cell, which cell is suspended in afluid comprising one or more ion selected from: Na⁺, K⁺, Cl⁻, H⁺, Ca²⁺,and HCO₃ ⁻.
 33. The method of claim 7 or 13, wherein the one or moremembrane is a cell membrane.
 34. The method of claim 33, wherein thecell membrane is present in an intact or live cell, or, wherein the cellis selected from: an animal cell, a plant cell, a fungal cell, abacterial cell, a mammalian cell, a primate cell, a rodent cell, acanine cell, a feline cell, a livestock cell, a cultured cell, a THP-1cell, a COS cell, a CHO cell, a HEK cell, a HeLA cell, an NIH 3T3 cell,a primary cell, an endoderm cell, an ectoderm cell, a mesoderm cell, aprimary cell derived from differentiated tissue, a primary cell derivedfrom undifferentiated tissue, a primary cell derived from blood, aprimary cell derived from peripheral blood, a primary cell derived fromnerve, a primary cell derived from muscle, a primary cell derived fromskin and a primary cell derived from bone.
 35. The method of claim 34,wherein the intact or live cell has a transmembrane potential of about−100 mV to about 10mV.
 36. The method of claim 7 or 13, wherein thefirst signal is detected optically.
 37. The method of claim 7 or 13,wherein the first signal is detected at between about 20° C. and 40° C.38. The method of claim 7 or 13, wherein monitoring the first signaloutput comprises detecting one or more fluorescent emission produced bythe cationic membrane permeable nucleic acid staining dye or themembrane permeable label.
 39. The method of claim 7 or 13, furthercomprising contacting the first component with a transmembrane potentialmodulator and monitoring an effect of the transmembrane potentialmodulator by monitoring the first signal output.
 40. The method of claim39, wherein the transmembrane potential modulator is a control modulatoror a test modulator.
 41. The method of claim 40, wherein the controlmodulator is selected from: a molecule, a neurotoxin, a set ofneurotoxins, a neurotransmitter, a set of neurotransmitters, a protein,a set of proteins, a peptide, a set of peptides, a lipid, a set oflipids, a carbohydrate, a set of carbohydrates, an organic molecule, aset of organic molecules, a drug, a set of drugs, a receptor ligand, aset of receptor ligands, an antibody, a set of antibodies, a cytokine, aset of cytokines, a chemokine, a set of chemokines, a hormone, a set ofhormones, a cell, a set of cells, a protein attached to a cell, and aprotein attached to a set of cells.
 42. A method of generating anoptical signal which is sensitive to transmembrane potential, the methodcomprising: providing a first component comprising one or more membrane;adding a cationic membrane permeable redistributing dye to the firstcomponent; adding an anionic membrane permeable redistributing dye tothe first component; and, measuring a first signal output from thecationic dye and a second signal output from the anionic dye, whereinone or more of the first and second signal outputs comprises an opticalsignal output, thereby generating the optical signal which is sensitiveto the transmembrane potential.
 43. The method of claim 42, wherein thecationic dye is a membrane permeable nucleic acid staining dye or acationic rhodamine, an indo-carbocyanine dye, a thio-carbocyanine dye,an oxa-carbocyanine dye, an amino napthylethylenyl pyridinium dye, adialkyl amino phenyl polyphenyl pyridinium dye, or wherein the anionicmembrane permeable redistributing dye comprises one or more of: OxonolV, Oxonol VI, and DiBAC₄(3) DiBAC₄(5), DiBAC₂(3).
 44. The method ofclaim 42, further comprising adding a neutral dye to the firstcomponent.
 45. The method of claim 42, further comprising adding aneutral dye to the first component, wherein the neutral dye produces acontrol signal output which is dependent on one or more of: temperature,incubation time and overall membrane permeability.
 46. A method ofgenerating an optical signal which is dependent on transmembranepotential, the method comprising: providing a first component comprisingone or more membrane; adding at least a first membrane permeableredistributing dye to the first component, wherein the first membranepermeable redistributing dye comprises an ion; measuring one or moresignal output from the first redistributing dye before an equilibriumdye distribution is established, which one or more signal outputcomprises at least one optical signal output, thereby providing theoptical signal which is dependent on transmembrane potential.
 47. Themethod of claim 46, further comprising correlating the one or moresignal output to a change in transmembrane potential.
 48. The method ofclaim 46, comprising adding at least a second membrane permeableredistributing dye to the one or more component and measuring one ormore signal outputs from the second membrane permeable redistributingdye before an equilibrium dye distribution is established.
 49. Themethod of claim 48, wherein the first and second dyes are added to thefirst component at approximately the same time and the signal outputsfrom the first and second membrane permeable redistributing dyes aremeasured at approximately the same time.
 50. The method of claim 48,wherein the first and second redistributing dyes comprise an anionic dyeand a cationic dye.
 51. The method of claim 48 or 50, wherein the firstand second redistributing dyes comprise one or more of: an anionic dye,a cationic dye, a cationic membrane permeable nucleic acid staining dye,and a neutral dye.
 52. The method of claim 48, further comprising addingat least a third membrane permeable redistributing dye to the one ormore component and measuring one or more signal outputs from the thirdmembrane permeable redistributing dye before an equilibrium dyedistribution is established.
 53. The method of claim 52, wherein thefirst and second redistributing dyes comprise one or more of: an anionicdye and a cationic dye and wherein the third membrane permeableredistributing dye comprises a neutral dye.
 54. The method of claim 53,wherein the signal output for the neutral dye is correlated to atemperature-dependent change in membrane permeability or atime-dependent change in membrane permeability.
 55. The method of claim51, wherein the cationic dye is a nucleic acid staining dye.
 56. Themethod of claim 51, wherein the cationic dye comprises a nucleic acidstaining dye selected from: a Blue-fluorescent SYTO dye, aGreen-fluorescent SYTO Dye, an Orange-fluorescent SYTO dye, aRed-fluorescent SYTO dye, Pur-1, thiazol, aryl, 2DS-7J1, Hoechst 33258,Hoechst 33342 and hexidium iodide or wherein the anionic redistributingdye comprises one or more of Oxonol V, Oxonol VI, and DiBAC₄(3)DiBAC₄(5), DiBAC₂(3).
 57. The method of claim 42, wherein the anionicredistributing dye comprises Syto 62 and the anionic dye comprisesDiBAC₄(3).
 58. A microfluidic device for monitoring transmembranepotential, the microfluidic device comprising: a body structure havingat least one microscale cavity disposed therein; a target source of afirst composition comprising at least one membrane, which target sourceis fluidly coupled to the at least one microscale cavity; and, a sourceof one or more voltage sensitive dyes which source is fluidly coupled tothe at least one microscale cavity, wherein, during operation of thedevice, the first composition is contacted to the one or more voltagesensitive dyes in the at least one microscale channel.
 59. The device ofclaim 58, wherein the one or more voltage sensitive dyes comprise one ormore of: a cationic membrane permeable staining dye source comprisingone or more cationic membrane permeable dyes, which cationic membranepermeable dye source is fluidly coupled to the at least one microscalecavity, and an anionic membrane permeable redistributing dye sourcecomprising one or more anionic redistributing dye, which anionicredistributing dye source is fluidly coupled to the at least onemicroscale cavity.
 60. The device of claim 58, wherein the cationic dyesource is a nucleic acid staining dye.
 61. The device of claim 58,wherein the device comprises both the cationic membrane permeablestaining dye source and the anionic membrane permeable redistributingdye source, and wherein, during operation of the device, the firstcomposition is contacted, in the presence of the cationic membranepermeable dye and the anionic membrane permeable redistributing dye, toat least one transmembrane potential modulatory composition.
 62. Thedevice of claim 58, the device further comprising a source of at leastone potential transmembrane potential modulatory composition, whichsource is fluidly coupled to the at least one microscale cavity.
 63. Thedevice of claim 58, further comprising a source of at least onetransmembrane potential modulatory composition, wherein, duringoperation of the device, at least one transmembrane potential modulatorycomposition is contacted to one or more of: the first composition, thecationic membrane permeable dye, or the anionic membrane permeableredistributing dye.
 64. The device of claim 58, the device furthercomprising a plurality of sources of at least one potential membranemodulatory composition.
 65. The device of claim 64, wherein theplurality of sources comprise one or more microtiter trays, each of theone or more trays comprising at least one potential membrane modulatorycomposition.
 66. The device of claim 65, wherein the microtiter traysare movably mounted proximal to the body structure, wherein the bodystructure comprises one or more pipettor channels which are structurallyconfigured to access the trays, which one or more pipettor channels arefluidly coupled to the at least one microscale cavity.
 67. The device ofclaim 62, wherein the at least one potential membrane modulatorycomposition comprises one or more of: a membrane hyperpolarizationbuffer, a membrane depolarization buffer, a compound which altersmembrane permeability and a compound which alters transport of an ionacross the cell membrane.
 68. The device of claim 58, further comprisinga signal detector located proximal to or within the microscale cavity,which signal detector detects the a signal.
 69. The device of claim 68,wherein the detector detects the detectable signal for a selected lengthof time (t), or a selected time point (t_(p)).
 70. The device of claim58, wherein the at least one microscale cavity is a first microscalechannel, and wherein, during operation of the device: the firstcomposition comprising the at least one membrane is flowed from thetarget source into the microchannel; the cationic membrane permeable dyeor the an anionic membrane permeable redistributing dye is flowed intocontact with the first composition; and, the detectable signal ismonitored at one or more selected time points after contact of the firstcomposition with the cationic membrane permeable dye or the an anionicmembrane permeable redistributing dye.
 71. The device of claim 70,wherein at least one potential membrane modulatory composition is flowedfrom the target source into contact with the first composition.
 72. Thedevice of claim 58 wherein the membrane is a component of an intactcell.